Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-[(1-methylethylidene)di-4,1-phenylene]bis[.omega.-hydroxy-, mixed acrylates and isononanoates

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2017 - 22 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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

Test material information

Constituent 1

Reference substance name:

Esterification products of 4,4'-Isopropylidenediphenol, ethoxylated and prop-2-enoic acid and 3,5,5-trimethylhexanoic acid

EC Number:

919-846-5

Cas Number:

1187203-83-3

Molecular formula:

Not available for this UVCB.

IUPAC Name:

Esterification products of 4,4'-Isopropylidenediphenol, ethoxylated and prop-2-enoic acid and 3,5,5-trimethylhexanoic acid

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: on the first day of treatment, the animals were approximately 6 weeks old
- Mean on the first day of treatment: the males had a mean body weight of 260 g (range: 231 g to 292 g) and the females had a mean body weight of 201 g (range: 168 g to 232 g)
- Fasting period before study: no
- Housing: the animals were housed in twos or threes, by sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for 13 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: 08 November 2017 to 22 March 2018.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on oral exposure:

PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection (HPLC/UV)
Test item concentrations: were within an acceptable range of vari ation compared to the nominal values (± 15%) in Weeks 1, 4, 8 and 13.
Homogeneity: homogeneous formulations at 1 and 200 mg/mL.
Stability: not assessed, dose formulation were prepared daily.

Duration of treatment / exposure:

13 weeks: followed by a 6-week treatment-free period

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex per dose
5 additional animals per sex in controls and high-dose (investigation of reversibility) and 8 additional animals (satellite) per sex and per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose selection:
The dose levels were selected by the Sponsor based on the results of a previous OECD 422 study.
In this study, the test item was administered daily by gavage to male and female Sprague-Dawley rats for 2 weeks before mating, during mating and, for females, throughout gestation and until Day 5 post-partum, at the dose level of 100, 300 or 1000 mg/kg/day.
There were no toxicologically important changes in any of the parental parameters (i.e. clinical signs, Functional Observation Battery, motor activity, body weight, food consumption, seminology or estrous cycle).
At clinical pathology, non-adverse test item related effects were noted at 300 and 1000 mg/kg/day (lower white blood cell count at 1000 mg/kg/day in males; shortened activated partial thromboplastin time and higher cholesterol levels at 300 and 1000 mg/kg/day in both sexes; higher urine volume and lower specific gravity at 1000 mg/kg/day in both sexes).
At histopathology investigations, the test item at 1000 mg/kg/day induced slight increases in liver weights and minimal or slight decreases in thymus weights in both sexes, and minimally increased kidney weights in males only. No test item-related macroscopic findings attributed to the test item administration were noted.
At microscopic examination, there was minimal centrilobular hepatocellular hypertrophy in the liver of some males and females at 1000 mg/kg/day, minimal hyperplasia with increased inflammation in the cecum of males from 100 mg/kg/day, decrease in the paracortex size/cellularity of the mesenteric lymph node in one male and one female at 300 mg/kg/day and in some males and females at 1000 mg/kg/day, and a marginal increase in the incidence of lymphoid atrophy in the thymus at 1000 mg/kg/day.
Renal microscopic findings consisted of hyaline droplet nephropathy from 100 mg/kg/day, associated with increased tubular basophilia at 300 and 1000 mg/kg/day (males) or minimal or slight tubular vacuolation from 100 mg/kg/day (females). These lesions were associated with increases in the urine volume and were considered as adverse at 1000 mg/kg/day.
The dose level of 300 mg/kg/day was therefore considered to be the No Observed Adverse Effect Level (NOAEL) for parental and systemic toxicity.

Thus, based on these available data, the selected dose levels of the present study were: 100, 300 and 1000 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, including weekends and public holidays.

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

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

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

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week (over 7-day periods) during the study.

WATER CONSUMPTION:
- Time schedule: The quantity of water consumed by the animals in each cage was recorded at least once a week (over 7 day periods) during the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: ophthalmological examinations were performed on all animals, before the beginning of the treatment period, on control- and high-dose principal animals on one occasion at the end of the treatment period (i.e. Week 13).
As no relevant changes were detected in the high-dose principal animals at the end of the treatment period, the recovery animals were not examined.

FUNCTIONAL OBSERVATION BATTERY (FOB) (principal and recovery animals):
Each principal animal was evaluated once in Week 12 before the daily treatment.
As no relevant changes were observed at the end of the treatment period, these examinations were not performed at the end of the treatment-free period.
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.

DETAILED CLINICAL OBERVATION:
The following parameters were assessed and graded:
- in the cage "touch escape",
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyper- activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.

Reactivity to manipulation and different stimuli
The following measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupil reflex,
- visual stimulus,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- at the end of observation: rectal temperature.

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

HAEMATOLOGY:
- Time schedule for collection of blood: the parameters were determined for all principal animals at the end of the treatment period (i.e. Week 13). In view of the findings observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: the parameters were determined for all principal animals at the end of the treatment period (i.e. Week 13). In view of the findings observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [2] were examined.

URINALYSIS:
- Time schedule for collection of urine: the parameters were determined for all principal animals at the end of the treatment period (i.e. Week 13). In view of the findings observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [3] were examined.

Sacrifice and pathology:

ORGAN WEIGHTS: see table below
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 principal and recovery animals.
No macroscopic post-mortem examination was performed on satellite animals euthanized at the end of the treatment period.

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

PREPARATION OF HISTOLOGICAL SLIDES (principal and recovery animals)
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert, et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin.
The kidneys of principal males in groups 1 and 4 euthanized at the end of the treatment period were immunostained with an antibody for a2u-globulin protein due to an indication of hyaline droplet accumulation at examination of the hematoxylin-eosin slides.

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

As required according to kidney histopathology, immunostained kidneys from control- and high-dose males (groups 1 and 4) euthanized at the end of the treatment period were examined.
In agreement with the Sponsor and based upon the microscopic results of the high-dose group at the end of the treatment period, the following tissues were examined for low- and intermediate-dose animals (groups 2 and 3) and recovery animals (groups 1 and 4):
- ileum,
- kidneys,
- liver,
- thyroids,
- thymus.

Other examinations:

EVALUATION OF T-CELL DEPENDANT ANTIBODY RESPONSE (TDAR) (satellite animals)
Immunization with KLH
On Days 77 and 84, 0.2 mL of KLH solution at 1.5 mg/mL was administered by subcutaneous injection in the interscapular area using a sterile plastic syringe fitted with a single-use sterile needle. KLH solution (total amount of 0.3 mg per injection) was administered before treatment.
The injection site was clipped using an electric clipper on the day before each administration and a circle was marked with an indelible pen (surgical pen) to show the approximate injection area. The injection site was disinfected with 70% alcohol prior to injection.

Quantification of anti-KLH IgM and IgG response
Venous blood (1 mL) was collected from the jugular vein of each animal into a plain tube at the following time points:
- once during the pre-treatment period (supernumerary animals included),
- on Day 84 (before any treatment),
- on Day 91 (before euthanasia).

The blood was kept at room temperature for 30 minutes to allow clotting (maximum 2 hours) and then centrifuged (at approximately 3000g for 10 minutes at +4°C). The resulting serum was split into four aliquots (3 x 75 µL and one with the remaining volume) in polypropylene tubes. The tubes were kept at 20°C until analysis.
Specific anti-KLH IgM and anti-KLH IgG levels were measured (using specific ELISA methods developed and validated at Citoxlab France) for all satellite animals. The results were expressed in terms of concentration.
Following collection of the last blood sample, the satellite animals were euthanized and discarded without any further examinations.

IMMUNOPHENOTYPING (principal and recovery animals)
Blood
Fasting was not necessary for the immunophenotyping analyses. However, as blood samples were collected at the same time for laboratory investigations (hematology and biochemistry) in Week 13, the animals were deprived of food for a period of at least 14 hours.
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.

Flow cytometry method
Objective
The immunophenotyping panel is based on the combination of six specific membrane markers (CD45; CD3; CD4; CD8; CD45RA and CD161a) to determine the relative and absolute counts of the following lymphocyte subsets:
- T cells (CD45+/CD3+),
- CD4 T cells (CD45+/CD3+/CD4+),
- CD8 T cells (CD45+/CD3+/CD8+),
- B cells (CD45+/CD3-/CD45RA+),
- NK cells (CD45+/CD3-/CD161a+),
- NKT cells (CD45+/CD3+/CD161a+).

SPERM PARAMETERS (principal and recovery animals)
These examinations were performed in all principal males.
In view of the findings observed at the end of the treatment period, testicular sperm count investigations were also carried out at the end of the treatment-free period.
Before euthanasia at the end of the treatment or treatment free-period period, each male was anesthetized by an intraperitoneal injection of sodium pentobarbital.

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

Epididymal sperm motility
The sperm was evaluated on a slide, after appropriate dilution if necessary. The numbers of motile and immotile spermatozoa from a sample of 200 spermatozoa were evaluated under a microscope using a 40 fold magnification. Results are expressed as the proportions 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 proportions 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 cauda of the left epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was collected and the number of spermatozoa was counted in a microscope slide-counting chamber.
Results are expressed as the numbers of spermatozoa per cauda and per gram of cauda.

Testicular sperm
The left testis was frozen at -20°C pending 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; Blazak et al., 1993).

MONITORING OF ESTROUS CYCLE (principal and recovery animals)
The estrous cycle stage was determined for each principal female from a fresh vaginal lavage (stained with methylene blue), daily for 21 consecutive days at the end of the treatment period.
As no relevant changes were observed at the end of the treatment period, these examinations were not performed at the end of the treatment-free period.

THYROID HORMONES
An additional blood sample was taken from each animal at the end of the treatment (principal animals in Week 13) and treatment-free (recovery animals) periods.

The levels of T4 thyroid hormone were determined by LC/MS-MS for all principal and recovery animals.

The levels of T3 thyroid hormone (determination by LC/MS-MS) and thyroid stimulating hormone (TSH, determination using a Luminex® method) were determined for all principal and recovery surviving animals in agreement with the Sponsor in view of the results obtained for T4 thyroid hormone.

Statistics:

Citox software was used to perform the statistical analysis of body weight, food consumption, water consumption, motor activity, anti-KLH IgM and IgG levels, lymphocyte populations, sperm parameters, thyroid hormones, 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 1.
Principal and recovery animals
Ptyalism was transiently observed in control males, and in males and females from all test item-treated groups in a dose-related manner, at the earliest from the first week of treatment in the high-dose group. This clinical sign was more marked in males and females from 300 mg/kg/day and was seen at the restraint just before administration in some of these animals.

Reflux at administration was noted in 3/15 males and 1/15 females given 0 mg/kg/day, 1/10 males given 100 mg/kg/day, 1/10 females given 300 mg/kg/day and 2/15 females given 1000 mg/kg/day on one to three occasions. This sign is commonly observed when a test item is administered by gavage and was therefore considered to be of no toxicological importance.

The other clinical signs recorded during the study, i.e., alopecia, scabs, thinning of hair, bent tail, soiled head, chromodacryorrhea, chromorhynorrhea, abnormal growth of teeth, hunched posture, piloerection and/or increase in size muzzle were of isolated occurrence, were observed both in control and test item treated animals, and/or had no dose-relationship. They were therefore considered to be unrelated to the test item.

Test item-related clinical signs were no longer observed in surviving animals over the treatment-free period.

Satellite animals
Similar clinical signs were reported in satellite animals.

Thickening and/or nodosities and/or scabs were noted at the injection site in all control and test item treated animals (with the exception of one control male). They were attributed to the intramuscular injection of KLH in the interscapular region on Days 84 and 91 for the immunological investigations.

Other clinical signs (hyperactivity and exophthalmos) were not attributed to the test item treatment or to the injection of KLH as they were observed only on Day 34 in one control female.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related deaths occurred during the study.
One control female was prematurely euthanized for humane reasons on Day 113 (Week 17). Before euthanasia, thin appearance, pallor of extremities, abdominal and loud breathing, chromodacryorrhea and abnormal growth of teeth were observed. A body weight loss of 47 g (-15% of the previous body weight) was recorded between Weeks 15 and 16, along with slightly reduced food consumption (16.4 g/animal/day for the cage vs. 19.8 g/animal/day over the previous week). At necropsy, a cranial mass with brain deformation and abnormal growth of teeth were noted. The cranial mass was a benign bone neoplasm (osteoma), with hemorrhage.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 3.
At 1000 mg/kg/day in males, when compared with controls, statistically significant lower mean body weight gain was recorded throughout the treatment period (-20% vs. controls) leading to a statistically significant lower mean body weight from Week 2 (-6% vs. controls) until the end of the treatment period (-13% vs. controls in Week 13). The differences in body weight gain were particularly noticeable over the second month of treatment (-22% vs. controls). During the recovery period, a statistically significant higher mean body weight gain was observed in previously treated males, leading to a terminal mean body weight slightly lower than that of the control animals (-8% vs. controls) without statistically significance. These effects were attributed to the test item and were considered as non-adverse in view of their magnitude and reversibility.
In females, the body weight was not affected by the test item although the body weight evolution fluctuated throughout the treatment period. The statistically significant lower mean body weight gain observed during the third month (-69%) was mainly due to a slightly mean body weight loss of 4 g recorded for the last week of treatment. This isolated and minimal difference was considered to be of no toxicological importance.


At 300 mg/kg/day in males, when compared with controls, statistically significant, lower mean body weight gain was recorded throughout the treatment period (-14% vs. controls) leading to a slightly lower mean body weight at the end of the treatment period (-7% vs. controls in Week 13). Instances of lower mean body weight gain were observed, particularly throughout the first month of the treatment period (-14% vs. control, p<0.01). These effects were attributed to the test item and were considered as non-adverse in view of their magnitude.
In females, body weight was not affected by the test item although body weight evolution fluctuated throughout the treatment period. A slight mean body weight loss of 2 g was recorded in the last week of the treatment period. This isolated and minimal difference was considered to be of no toxicological importance.

At 100 mg/kg/day in males and females, when compared with controls, no relevant effects were noted on body weight or body weight gain during the treatment period. Some instances of lower mean body weight gain were observed for males in Week 9 (-29%, p<0.05) and some instances of higher mean body weight gain were observed for females in Weeks 4 and 6 (+89% and +117% respectively, p<0.01). These sporadic variations without relevant effects on the terminal mean body weight and of opposite trends were considered to be of no toxicological importance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Table 4.
At 1000 mg/kg/day in males, when compared with controls, statistically significant lower mean food consumption, was noted over the first month of the treatment period and in Week 6 (-9% to -13%) only. Throughout this period, this correlated with the lower mean body weight gain observed in males at this dose level. No relevant differences from controls were recorded in the mean food consumption of previously test item treated males during the treatment-free period.

At 300 mg/kg/day in males, when compared with controls, statistically significant lower mean food consumption was noted in Week 4 of the treatment period (-9%) only.

Effects observed in males given 300 or 1000 mg/kg/day were attributed to the test item but were considered as non-adverse in view of their magnitude, their isolated or limited occurrence and/or their reversibility.

No relevant effects were observed on food consumption in test item-treated females during the treatment or treatment-free period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

See Table 5.
At 1000 mg/kg/day and when compared with controls, statistically significant, moderately to markedly, higher mean water consumption was recorded from the second week of the study until the end of the treatment period in males (+13% to +57%; p<0.01) and over the whole treatment period in females (+29 to +99%; p<0.01). These differences were more marked in females, increased over time and persisted in previously test item-treated males (+17% to +78%) and females (+8% to +104%) during the treatment free period, but to a lesser extent as they were not statistically significant and tended to return to normal values at the end of the treatment-free period. These test item-related findings correlated with non-adverse slightly higher mean urine volumes at the end of the treatment period in males and females given 1000 mg/kg/day. They were considered as non-adverse in view of their partial recovery and as they were not associated with any adverse effects.

At 300 mg/kg/day in males, when compared with controls, sporadic, statistically significant, higher mean water consumption was noted in Weeks 5, 7, 11 and 12 of the treatment period (+14% to +19%; p<0.05 to p<0.01). This effect was attributed to the test item but considered as non-adverse in view of the low magnitude and isolated occurrence.

Other instances of differences from controls were noted during the treatment period, namely in males or females given 100 mg/kg/day in Week 5 or 9 (males: +16% and females: +17%, respectively; p<0.01) and in females given 300 mg/kg/day in Week 9 (+14%; p<0.05). As these variations were not dose-related, of isolated occurrence and/or resulted from lower mean water consumption in controls, they were considered to be fortuitous and not test item-related.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related findings were observed in principal animals at the end of the treatment period.

Chorioretinopathy was noted in 1/10 principal females given 1000 mg/kg/day in week 13. As this finding is commonly observed in rats of this strain and age and as it was noted in a single animal, it was considered to be fortuitous and not test item-related.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 7.
End of treatment period
At 1000 mg/kg/day in males and females, when compared with controls, minimal lower red blood cell parameters, including mean red blood cell count (males only: -7%; p<0.01), mean hemoglobin concentration (males: -5% and females: -6%; p<0.01) and mean packed cell volume (males: -6% and females: -4%; p<0.01) were noted. Minimal decrease in reticulocyte count was also observed in males (-19%; p<0.01) and females (-16%; p<0.05). These effects were considered to be test item related but of minor toxicological importance in the absence of correlates with any histopathological findings, as they were of minimal magnitude, as values were close to those recorded in recovery control animals and/or remained within the range of the Historical Control Data.
At this dose level, in males, minimally lower mean white blood cell count (-18%; p<0.05), associated with lower mean eosinophil count (-45%; p<0.01), were observed along with lower mean lymphocyte count (-20%; p<0.05). Shortened prothrombin time was recorded in males (-8%; p<0.01) and to a lesser extent in females (-5%; p<0.05). Lower white blood cell counts correlated with a non-adverse decrease of B cells subset and a non-adverse minimal lymphoid atrophy of the thymus. These effects were considered to be test item-related and of minor toxicological importance as values remained within the range of the Historical Control Data.

At 300 mg/kg/day in males, when compared with controls, similar but slightly less pronounced differences were noted in red blood cell parameters, including mean red blood cell count (-4%; p<0.05), mean hemoglobin concentration (-4%; p<0.05), mean packed cell volume (-4%; p<0.05) and reticulocyte count (-10%; p<0.05). These effects were considered to be of no toxicological importance, as they were of minimal magnitude, did not correlate with any histopathological findings, as values were close to those recorded in recovery control animals and/or as values remained within the range of Historical Control Data.

The other differences between control and test item-treated animals at the end of the treatment period, namely in reticulocyte count (females given 100 mg/kg/day) or in packed cell volume (males given 100 mg/kg/day) were considered to be of no toxicological importance as they were of low magnitude, within the range of the Historical Control Data, noted with no dose-relationship and/or without any correlating findings.

End of treatment-free period
When compared with controls, lower mean reticulocyte count was still observed in males previously given 1000 mg/kg/day (-19%, not statistically significant). This difference was noted with a lower magnitude than at the end of the treatment period and the mean value was similar to the control mean value recorded at the end of the treatment period, suggesting a a full-recovery for this parameter.
The other findings were no longer observed in recovery animals over the treatment-free period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 8.
End of treatment period
When compared with mean control values, relevant blood biochemistry changes were noted:
- lower mean potassium level in females at 100, 300 and 1000 mg/kg/day (-7%, -8% and -6%, respectively; p<0.05 or p<0.01) and males at 1000 mg/kg/day (-5%; p<0.05),
- higher mean inorganic phosphorus level in females at 1000 mg/kg/day (+9%; p<0.05),
- lower mean glucose level in males and females at 1000 mg/kg/day (-10% with p<0.01 in males only),
- higher mean urea level in males at 300 mg/kg/day (+15%; p<0.05) and in males and females at 1000 mg/kg/day (+18% and +34%, respectively; p<0.01) associated with higher mean creatinine level in males and females at 1000 mg/kg/day (+9% and +15% with p<0.01, respectively),
- higher mean protein level in females at 100, 300 and 1000 mg/kg/day (+11%, +9% and +10%, respectively; p<0.05 or p<0.01) and in males at 1000 mg/kg/day (+3%; p<0.05) along with higher mean calcium level in males and females at 300 mg/kg/day (+3% and +5%, respectively; p<0.01) and 1000 mg/kg/day (+6% and +13%, respectively; p<0.01),
- higher mean albumin level in females at all dose levels (+8%; p<0.01) and lower mean albumin/globulin ratio level (-5%; p<0.05) in males at 1000 mg/kg/day.

These changes in biochemistry parameters were more marked in females. Although some of these findings were above the upper limit of the Historical Control Data (namely calcium in both sexes at 1000 mg/kg/day), effects on potassium, calcium, inorganic phosphorus, glucose, protein, albumin, creatinine and urea levels were considered to be of minor toxicological importance, as they were of minimal magnitude, poorly dose-related, reversible and/or did not correlate with histopathological findings (hyaline droplet nephropathy associated with granular casts and focal tubular basophilia was observed in males only). It may be noted that one female given 1000 mg/kg/day having the highest urea level (7.9 mmol/L) also showed moderate pyelonephritis.

The other statistically significant differences observed between control and test item-treated animals consisted of higher mean cholesterol level in males and females at 100 (+42% and +47%, respectively; p<0.01), 300 (+55% and +88%, respectively; p<0.01) and 1000 mg/kg/day (2.5 and 2.0 fold, respectively; p<0.01), together with higher mean alkaline phosphatase activity in males at 1000 mg/kg/day (+34%; p<0.05). These differences were dose-related and above the upper limit of the Historical Control Data for most of them. As they were reversible and had no relationship with adverse microscopic findings, they were considered to be of minor toxicological importance.

End of treatment-free period
Statistically significant, higher mean urea level (+19% vs. controls; p<0.01) was still observed in males previously given 1000 mg/kg/day. As this difference was of minimal magnitude, closed to the upper limit of the Historical Control Data, not linked to the renal tubular basophilia (ongoing almost complete recovery) and remained at the same severity than that observed at the end of the treatment period, this was considered not to be adverse.

Higher mean inorganic phosphorus (+18% vs. controls; p<0.05) and chloride (+2% vs. controls; p<0.05) levels observed in females and males previously given 1000 mg/kg/day, respectively, and lower mean triglyceride level (-33% vs. controls; p<0.05) in females at the same dose level were considered to be unrelated to the test item as the differences were below the mean control values recorded at the end of the treatment period, and they were not observed at the end of the treatment period and/or they remained within the range of physiological values.

Reversibility of the other findings was noted at the end of the treatment-free period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 10.
End of treatment period
At 1000 mg/kg/day in males and females, when compared with controls, statistically significant higher mean urinary volumes were noted (2-fold and 2.2-fold, respectively; p<0.01) along with a tendency towards a slight decrease of specific gravity in females (-1%; p<0.05) or a lower mean pH in males (-13%; p<0.01). These differences were of low magnitude, close to the values recorded in controls at the end of the treatment-free period, within the range of the Historical Control Data and/or reversible, therefore they were considered to be of minor toxicological importance.

Other differences from controls were observed:
- presence of a few to several calcium oxalate crystals in all fields in 7/10 females at 300 mg/kg/day and 8/10 males and 9/10 females at 1000 mg/kg/day. This correlated with higher mean calcium level in blood at the same dose levels,
- presence of ketones (= 3.9 mmol/L) in 4/10 males given 1000 mg/kg/day,
- absence of proteins in 9/10 females at 300 mg/kg/day and in 10/10 males and 9/10 females at 1000 mg/kg/day, absence of bilirubin in 9/10 females and 10/10 males at 1000 mg/kg/day and absence of magnesium ammonium phosphate crystals in 8/10 males at 1000 mg/kg/day. Given the direction of the changes, and as there were no correlations with any other findings, these differences were considered to be of no toxicological importance,
- presence of several leucocytes in all fields in 2/10 females at 1000 mg/kg/day. This corresponded to incidental pyelonephritis in two females, and consequently was considered not to be test item-related.

End of treatment-free period
Test item-related effects on the urinary parameters were no longer observed in animals given 1000 mg/kg/day at the end of the treatment-free period.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

See Table 2.
There were non adverse test item-related effects on functional observation battery tests or motor activity data in any group.

Differences from controls were noted in the mean number of horizontal movements (i.e. -11% in males given 100 mg/kg/day and -16% in females given 300 mg/kg/day) and rearing movements (i.e. -13% in males given 1000 mg/kg/day). They were considered to be of no toxicological importance as they were of minor magnitude (not statistically significant), poorly dose-related and/or as values remained close to or within the standard deviation of control values.

Absence of grooming was noted in 10/10 males given 300 or 1000 mg/kg/day vs. 8/10 control males. In view of the very slight magnitude, and in the absence of correlating clinical signs during the study, this finding was considered to be unrelated to the test item treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See Table 15.
End of treatment period
The mean body weight at necropsy was decreased in high-dose males. Many minor differences in organ weights in this group compared to controls were the direct consequence of this lower final body weight, and even though they reached on occasions statistical significance (for instance brain weight) they were unrelated to test item administration.

There were test item related differences in adrenal, liver, thyroid, kidneys and thymus weights.

Liver weights were increased at all doses in both sexes. The increase was dose-related. At the high dose in both sexes and at the mid-dose in females, it correlated histologically with centrilobular hypertrophy.

Non-statistically significant minimal increase in thyroid weight relative to body weight was present in males at the high-dose. It correlated microscopically with follicular cell hypertrophy in the thyroid.

Kidneys weights were minimally increased in both sexes at the high-dose. In males, it was associated with increased tubular hyaline droplets, basophilia and hyaline casts at histological examination.

Thymus weights were mildly decreased in both sexes at the high-dose. It correlated microscopically with minimal lymphoid atrophy.

Minimally higher adrenal weight relative to body weight in males at the high-dose did not have histological correlates. These differences in adrenal weights, along with thymic changes, were considered possibly related to a non-specific stress response to the test item.

All other differences between control and test item treated groups were considered to be related to individual variations because of their small amplitude, their lack of histological correlate and/or absence of dose relationship.

End of recovery period
At the end of the treatment-free period, final body weights were minimally decreased in males previously treated at the high-dose. Minor differences between controls and high-doses on the organ weights were considered to be the consequence of this decrease in terminal body weight, with no relationship to test item.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

End of recovery period
At the end of recovery all macroscopic observations were spontaneous and bore no relationship to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Tables 16 and 17.
End of treatment period
There were treatment-related changes in the ileum, kidneys (males only), liver and thyroids.
A dose related minimal to moderate increase in hyaline droplets in the kidneys was diagnosed in males at all doses. Hyaline droplets were composed of a2u-globulin, as confirmed by Immuno-Histo Chemistry (IHC) at the high-dose. The average score for IHC positivity in high-dose rats was 2.2, vs. 1.4 in controls, confirming the increase noted in hematoxylin and eosin-stained slides. Hyaline droplets were associated with granular casts and focal tubular basophilia (all doses) and mineralization at the cortico medullary junction (high-dose). Together, these findings, which were directly related to the test item, are part of the so-called a2u-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 granular casts, it was considered adverse from the dose of 300 mg/kg/day. In addition, mineralization at the cortico-medullary junction was observed in the high-dose males.

Renal inflammation (pyelitis or pyelonephritis) was observed in 4/10 females at 100 mg/kg/day and in 3/10 females at 1000 mg/kg/day. In some animals, it was associated with urothelial hyperplasia in the urinary bladder, suggesting an ascending origin of the inflammation. Ascending urinary inflammation can be observed spontaneously, particularly in females. In the present study, given its absence at the mid-dose and the absence of a difference in severity and incidence between the low- and the high-dose, these inflammations are considered spontaneous and unrelated to test item administration.

Minimal to slight centrilobular hypertrophy in the liver was observed at the high-dose (both sexes) and in females at the mid-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. At the high-dose, it was accompanied by minimal follicular cell hypertrophy of the thyroid. Follicular cell hypertrophy in the thyroid often accompanies hepatic centrilobular hypertrophy in rats, although this is not relevant to humans.

Vacuolation of Kupffer cells was observed in the liver in both sexes at the high-dose only. The origin of this change is unknown. Because of the macrophagic activity of these cells, it possibly represents accumulation of the test item or one of its metabolites. In the absence of associated clinical pathology changes, it is not considered as adverse.

Minimal to slight dilatation of the lacteals in the ileum (i.e. lymphangectasia) was observed in many animals at the high-dose (both sexes) and in 1/10 female at the mid-dose. This localized change within the digestive tract, of unknown origin, was unaccompanied by degenerative of inflammatory changes. It is considered non adverse.

Minimal thymic cortical atrophy was observed at the high-dose (both sexes).

The qualitative assessment of the bone marrow did not show any difference between test item-treated and control groups.

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

End of recovery period
At the end of the treatment-free period, minimal to slight renal tubular basophilia (males only), slight pyelitis (one female), minimal dilatation of lacteals in the ileum (one female only) and minimal cortical atrophy in the thymus (one male only) were observed in previously test item treated animals.

Like at the end of the treatment period, pyelitis is considered spontaneous.

A low incidence of minimal cortical atrophy of the thymus can be observed in controls and is considered spontaneous.

Renal tubular basophilia and dilatation of lacteals in the ileum, although considered test item related, demonstrate a much lower incidence and severity compared with end of treatment, indicating ongoing, almost complete, recovery.

Other changes considered test item related at the end of treatment were not observed, indicating full recovery.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Estrus cycle
See Table 6.
There were no test item-related effects on the mean length of the estrus cycle or the mean number of cycles.
A trend towards an increase in the mean length of diestrus was observed in females at all dose levels at the end of the treatment period. This effect was poorly dose-related and was considered to be of no toxicological importance in the absence of statistical significance or any corresponding microscopic findings.

Thyroid hormones
See Table 9.
End of treatment period
At 1000 mg/kg/day, when compared with controls, there were slightly to moderately increased mean T3 concentrations in males (+32%; p<0.01) and females (+22%), moderately increased mean T4 concentration in females (+33%; p<0.01), and severe increased mean TSH concentrations in males (+200%; p<0.01) and females (+197%; p<0.01).
These results were consistent with minimal thyroid cell hypertrophy observed at microscopic examination in 7/10 males and 7/10 females treated at 1000 mg/kg/day and induced by a non-adverse increased hepatic metabolism. These differences were considered to be test item-related and non-adverse as they were not associated with any adverse findings.

End of treatment-free period
The findings were no longer observed in recovery animals over the treatment-free period with the exception of mean T3 levels which were slighltly higher than controls in females previously given 1000 mg/kg/day suggesting ongoing recovery.

Determination of serum levels of anti-KLH IgM antibodies
See Table 11.
At baseline, background signals with OD values corresponding to quantifiable concentrations of anti-KLH IgM were detected in all samples. This result can be explained by the fact that rat serum is expected to contain IgMs directed against some glycosylated moieties similar to the ones of KLH. As animals were not exposed to KLH before immunization, it is unlikely that this corresponds to anti-KLH IgM.
Seven days after the first KLH injection (primary response, Day 84), anti-KLH IgM production was observed in all groups with fold changes ranging from 4.4 to 10.0.
Seven days after the second KLH injection, anti-KLH IgM concentration increased in all groups with fold changes ranging from 11.7 to 16.7.
A high inter individual variability was observed, especially post immunization showing that the immune response magnitude was individual dependant.
Nevertheless, there were no statistically significant and/or dose-related differences from controls suggesting any toxic effects on the immune system on the antibody response to KLH.

Determination of serum levels of anti-KLH IgG antibodies
See Table 12.
At baseline, sera samples did not contain quantifiable concentrations of anti-KLH IgG (BLQ).
Seven days after the first KLH injection (Day 84), a slight anti-KLH IgG production was observed in all groups with fold changes ranging from 1.0. to 3.5.
Seven days after the second KLH injection (Day 91), anti-KLH IgG concentration increased in all groups with fold changes ranging from 8.4 to 24.9.
Inter individual variability was observed, especially after the second immunization showing that the immune response magnitude was individual dependant.
Nevertheless, there were no statistically significant and/or dose-related differences from controls suggesting any toxic effects on the immune system on the antibody response to KLH.

Immunophenotyping
See Table 13.
End of treatment period
When compared with controls, decreased B cell subsets (in absolute counts) were observed in males given 300 or 1000 mg/kg/day (-24.6% and -35.2%, respectively; p<0.05 and p<0.01). A similar statistically significant result was found in relative counts in males given 1000 mg/kg/day (-18.5%; p<0.05). A similar tendency, even though not statistically significant, was observed in the females, both in absolute and relative counts (-20.8% and -16.6%, respectively), at 1000 mg/kg/day.
As these differences were statistically significant and dose-related, they were considered to be test item related and non-adverse in view of their magnitude, their correlation with non-adverse lymphoid atrophy and/or their reversibility.


End of treatment-free period
Changes in B cells subsets observed after the 13 weeks of treatment had completely recovered as they were no longer observed in recovery animals at the end of the treatment-free period.

When compared with controls, statistically significant mild decrease in cytotoxic T cells (CD8+ T cells) absolute count (-20.5%; p<0.05) was noted in males previously given 1000 mg/kg/day. However, individual values measured at the end of the treatment-free period in high-dose males (889 1261 cytotoxic T cells/µL) were within the range of values recorded in control animals (927-1430 cytotoxic T cells/µL). Furthermore no difference between treated and control groups was found in females. Therefore, the lower mean of cytotoxic T cells absolute count was considered to have mainly resulted from inter-individual variability, and not to be related to the test item administration.

Seminology
See Table 14.
No test item-related effects were noted on epididymal sperm count, motility or morphology.

Statistically significant lower mean values were recorded in the testicular sperm count of males from 100 mg/kg/day (-17% to -19% vs. controls; p<0.05). These findings were no longer observed in recovery animals at the end of the treatment-free period.
As these test item-related differences were poorly dose-related, of minor magnitude, without any microscopic correlates, and reversible, they were considered to be non-adverse.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Table 1: Clinical signs

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Ptyalism	2	8	10	15	-	5	10	15
Total affected animals	2/15	8/10	10/10	15/15	0/15	5/10	10/10	15/15

-: no clinical signs.

 

Table 2: Functional Observation Battery

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Horizontal movements	461	410	427	477	572	592	481	612
% from controls	-	-11	-7	+3	-	+3	-16	+7
Rearing	118	122	118	103	126	128	125	135
% from controls	-	+3	0	-13	-	+2	-1	+7

-: not applicable.

Table 3: Body weight and body weight change

Sex		Male					Female
Dose level (mg/kg/day)	0		100	300	1000	0		100	300	1000
Treatment period (principal and recovery animals)
Mean BW gain Weeks 1/5	+199		+196	+172**	+159**	+75		+89*	+71	+83
Mean BW gain Weeks 5/10	+116		+111	+104	+91**	+36		+40	+38	+39
Mean BW gain Weeks 10/13	+35		+29	+26	+30	+13		+9	+6	+4**
Mean BW gain Weeks 1/13	+350		+336	+302**	+280**	+124		+138	+114	+126
% from controls	-		-4	-14	-20	-		+11	-8	+2
Mean body weight Week 1	261		263	266	254	207		202	206	203
Mean body weight Week 2	325		326	325	306*	232		229	228	227
Mean body weight Week 3	382		384	373	351**	253		255	247	251
Mean body weight Week 5	460		460	438	413**	282		291	277	286
Mean body weight Week 13	612		600	569	535**	331		340	321	329
% from controls	-		-2	-7	-13	-		+3	-3	-1
Treatment-free period (recovery animals)
Mean BW gain Weeks 13/19	+41		-	-	+64*	+7		-	-	+5
Mean BW Week 19	653		-	-	600	349		-	-	329
% from controls	-		-	-	-8	-		-	-	-6

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

Table 4: Food consumption

 

	Sex		Male					Female
Dose level (mg/kg/day)		0		100	300	1000	0		100	300	1000
Treatment period (principal and recovery animals)
Week 1		27.2		27.9	27.4	24.2**	19.9		19.2	18.3	18.3
Week 2		29.6		30.5	28.1	26.8*	19.4		19.0	18.3	20.1
Week 3		28.4		28.5	26.5	25.7**	20.1		18.7	17.3	19.9
Week 4		27.9		28.1	25.3*	24.9*	18.0		20.1	17.5	19.8
Week 6		26.1		25.9	24.4	22.8**	17.6		18.1	16.4	17.6
Week 13		20.9		19.8	18.7	20.8	15.6		14.0	13.2	15.1

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

Table 5: Water consumption

 

	Sex		Male					Female
Dose level (mg/kg/day)		0		100	300	1000	0		100	300	1000
Treatment period (principal and recovery animals)
Week 1		41.6		43.3	40.8	42.3	31.3		33.3	31.4	40.3**
Week 2		39.2		41.5	41.5	48.4**	30.1		32.0	32.5	41.8**
Week 3		40.7		41.4	40.7	46.1**	28.2		31.4	30.1	41.4**
Week 4		38.7		41.4	40.6	47.1**	29.2		32.5	32.1	46.3**
Week 5		38.0		43.9**	43.2*	48.3**	29.9		32.7	32.3	45.3**
Week 6		37.5		40.8	41.9	49.5**	27.4		31.9	31.5	48.9**
Week 7		36.1		40.7	42.6**	50.0**	28.7		30.6	31.5	50.0**
Week 8		37.8		41.3	40.6	55.5**	30.3		33.7	33.7	51.5**
Week 9		36.5		40.4	40.8	51.3**	27.6		32.2**	31.5*	53.8**
Week 10		35.7		40.2	41.0	51.2**	28.8		31.6	33.4	57.3**
Week 11		34.3		37.6	40.8*	53.4**	31.7		35.1	35.3	56.0**
Week 12		34.2		37.4	40.1*	51.0**	28.7		33.6	32.9	55.0**
Week 13		31.5		35.0	35.0	49.4**	29.5		32.3	33.8	52.1**
Treatment-free period (recovery animals)
Week 14		35.4		-	-	49.2	28.4		-	-	49.4
Week 15		34.1		-	-	43.3	29.5		-	-	37.9
Week 16		35.8		-	-	46.0	29.8		-	-	37.5
Week 17		36.2		-	-	43.1	33.1		-	-	35.7
Week 18		37.0		-	-	42.2	33.6		-	-	40.3
Week 19		36.2		-	-	42.4	32.4		-	-	35.0

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

Table 6: Estrus cycle

Dose level (mg/kg/day)	0	100	300	1000
Treatment period (principal animals)
Number of cycles	3.9	3.8	3.1	3.8
Cycle length (days)	4.2	4.3	4.6	4.3
Number of females having a mean average cycle of 4-5 days	9	7	8	9

Table 7: Hematology

Sex	Male
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
	End of treatment period				End of treatment-free period
White blood cell count (G/L)	14.67 (6.08-25.95)[DP1]	13.54 -8%	13.07 -11%	12.10* -18%	13.44	-	-	13.78 +3%
Red blood cell count (T/L)	9.79 (8.49-10.33)[DP2]	9.43 -4%	9.37* -4%	9.10** -7%	9.54	-	-	9.16 -4%
Hemoglobin (g/dL)	16.9 (14.6-17.8)	16.3 -4%	16.2* -4%	16.0** -5%	15.7	-	-	15.6 -1%
Packed cell volume (L/L)	0.50 (0.44-0.54)	0.48* -4%	0.48* -4%	0.47** -6%	0.49	-	-	0.48 -2%
Reticulocyte count (T/L)	0.21 (-)	0.21 0%	0.19* -10%	0.17** -19%	0.27	-	-	0.22 -19%
Eosinophil count (G/L)	0.20 (0.00-0.37)	0.16 -20%	0.16 -20%	0.11** -45%	0.20	-	-	0.18 -10%
Lymphocyte count (G/L)	12.17 (5.10-22.73)[DP3]	11.22 -8%	10.64 -13%	9.72* -20%	10.48	-	-	10.98 +5%
Prothrombin time (s)	24.2 (20.7-27.0)	23.2 -4%	23.7 -2%	22.3** -8%	26.1	-	-	25.7 -2%

Statistically significant: *: p<0.05 and **: p<0.01.

In brackets, minimum-maximum values from Historical Control Data (HCD).

In italic, %vs. controls.

-: not applicable.

Sex	Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
	End of treatment period				End of treatment-free period
White blood cell count (G/L)	11.46 (3.18-19.81)	11.30 -1%	11.05 -4%	10.80 -6%	8.58	-	-	9.92 +16%
Red blood cell count (T/L)	8.48 (7.15-9.34)	8.56 +1%	8.32 -2%	8.11 -4%	8.61	-	-	8.37 -3%
Hemoglobin (g/dL)	14.9 (13.3-16.0)	14.8 -1%	14.7 -1%	14.0** -6%	15.1	-	-	14.7 -3%
Packed cell volume (L/L)	0.45 (0.39-0.50)	0.45 0%	0.45 0%	0.43** -4%	0.45	-	-	0.44 -2%
Reticulocyte count (T/L)	0.19 (-)	0.16* -16%	0.17 -11%	0.16* -16%	0.18	-	-	0.21 +17%
Eosinophil count (G/L)	0.13 (0.04-0.37)	0.19 +46%	0.13 0%	0.10 -23%	0.10	-	-	0.13 +30%
Lymphocyte count (G/L)	9.44 (2.48-16.99)	8.85 -6%	9.21 -2%	8.87 -6%	6.91	-	-	7.72 +12%
Prothrombin time (s)	26.1 (24.9-28.9)	25.4 -3%	25.1 -4%	24.7* -5%	27.0	-	-	27.5 +2%

Statistically significant: *: p<0.05 and **: p<0.01.

In brackets, minimum-maximum values from Historical Control Data (HCD).

In italic, %vs. controls.

-: not applicable.

Table 8: Blood biochemistry

Sex	Male
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
	End of treatment period				End of treatment-free period
Potassium (mmol/L)	3.92 (3.54-5.03)	4.01 +2%	3.97 +1%	3.72* -5%	3.77 (-)	-	-	3.96 +5%
Calcium (mmol/L)	2.70 (2.49-2.77)	2.70 0%	2.78** +3%	2.86** +6%	2.65 (-)	-	-	2.62 -1%
Inorganic phosphorus (mmol/L)	1.97 (1.72-2.29)	2.01 +2%	2.09 +6%	2.10 +7%	1.90 (-)	-	-	2.02 +6%
Glucose (mmol/L)	7.28 (5.17-9.92)	7.40 +2%	7.55 +4%	6.54** -10%	7.43 (-)	-	-	7.52 +1%
Urea (mmol/L)	4.0 (2.6-5.3)	4.0 0%	4.6* +15%	4.7** +18%	4.7 (-)	-	-	5.6** +19%
Creatinine (µmol/L)	37.24 (21.41-48.81)	36.56 -2%	37.79 +1%	40.59 +9%	40.84 (-)	-	-	41.25 +1%
Proteins (g/L)	59.4 (54.5-69.3)	59.4 0%	59.0 -1%	61.3* +3%	63.1 (-)	-	-	60.2 -5%
Albumin (g/L)	36 (33-41)	36 0%	36 0%	37 +3%	35 (-)	-	-	34 -3%
Albumin/globulin ratio	1.59 (1.28-1.81)	1.55 -3%	1.54 -3%	1.51* -5%	1.29  (-)	-	-	1.32 +2%
Cholesterol (mmol/L)	2.09 (0.98-2.54)	2.97** +42%	3.24** +55%	5.23** x 2.5	2.69 (-)	-	-	2.37 -12%
Alkaline phosphatase (U/L)	256 (166-601)	280 +9%	268 +5%	342* +34%	204 (-)	-	-	183 -10%

Statistically significant: *: p<0.05 and **: p<0.01.

In brackets, minimum-maximum values from Historical Control Data (HCD).

In italic, %vs. controls.

-: not applicable.

 

Sex	Female
Dose level (mg/kg/day)	0[DP4]	100	300	1000	0	100	300	1000
	End of treatment period				End of treatment-free period
Potassium (mmol/L)	3.87 (2.94-4.65)	3.60** -7%	3.55** -8%	3.64* -6%	3.34 (-)	-	-	3.66 +10%
Calcium (mmol/L)	2.66 (2.41-2.82)	2.71 +2%	2.78** +5%	3.00** +13%	2.60 (-)	-	-	2.60 0%
Inorganic phosphorus (mmol/L)	1.81 (1.30-2.18)	1.79 -1%	1.86 +3%	1.97* +9%	1.49 (-)	-	-	1.76* +18%
Glucose (mmol/L)	7.30 (4.91-9.41)	6.95 -5%	7.10 -3%	6.59 -10%	8.47 (-)	-	-	6.63 -22%
Urea (mmol/L)	4.4 (2.6-6.3)	4.9 +11%	4.5 +2%	5.9** +34%	5.7 (-)	-	-	5.5 -4%
Creatinine (µmol/L)	37.03 (27.11-46.93)	39.54 +7%	35.98 -3%	42.75** +15%	40.52 (-)	-	-	41.24 +2%
Proteins (g/L)	60.7 (55.7-80.1)	67.2* +11%	66.3** +9%	66.6** +10%	62.1 (-)	-	-	63.8 +3%
Albumin (g/L)	36 (33-50)	39** +8%	39** +8%	39** +8%	35 (-)	-	-	36 +3%
Albumin/globulin ratio	1.44 (1.18-1.87)	1.42 -1%	1.47 +2%	1.41 -2%	1.29  (-)	-	-	1.28 -1%
Cholesterol (mmol/L)	2.19 (1.10-3.05)	3.22** +47%	4.11** +88%	6.43** x 2.0	2.52 (-)	-	-	2.06 -18%
Alkaline phosphatase (U/L)	175 (73-468)	154 -12%	181 +3%	151 -14%	113 (-)	-	-	148 +31%

Statistically significant: *: p<0.05 and **: p<0.01.

In brackets, minimum-maximum values from Historical Control Data (HCD).

In italic, % vs. controls.

-: not applicable.

Table 9: Thyroid hormones

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
End of treatment period (principal animals)
T3 (ng/mL)	0.37 -	0.42 +14%	0.43 +16%	0.49** +32%	0.46 -	0.44 -4%	0.48 +4%	0.56 +22%
T4 (ng/mL)	38.66 -	41.91 +8%	42.65 +10%	37.74 -2%	30.61 -	31.30 +2%	36.59 +20%	40.70** +33%
TSH (ng/mL)	1486 -	2290 +54%	2266 +52%	4461** +200%	653 -	662 +1%	572 -12%	1941** +197%
End of treatment-free period (recovery animals)
T3 (ng/mL)	0.33 -	-	-	0.36 +9%	0.42 -	-	-	0.54 +29%
T4 (ng/mL)	36.42 -	-	-	34.92 -4%	27.95 -	-	-	32.01 +15%
TSH (ng/mL)	3401 -	-	-	1980 -42%	651 -	-	-	641 -2%

Statistically significant from controls:**: p<0.01

No Historical Control Data available.

In italic, % vs. controls.

-: not applicable.

Table 10: Urinalysis

	Urinary parameters
Sex	Male
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
	End of treatment period				End of treatment-free period
Volume (mL)	6 (2-17)	8 +33%	7 +17%	12** x2	11 (-)	-	-	12 +9%
Specify gravity	1039 (1025-1050)	1033 -1%	1033 -1%	1031 -1%	1031 (-)	-	-	1028 0%
pH	7.1 (6.0-8.0)	6.9 -3%	6.9 -3%	6.2** -13%	7.3 (-)	-	-	7.6 +4%

Statistically significant: **: p<0.01.

In brackets, minimum-maximum values vs. Historical Control Data (HCD).

In italic: % vs. controls.

-: not applicable.

	Urinary parameters
Sex	Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
	End of treatment period				End of treatment-free period
Volume (mL)	5 (2-21)	5 0%	5 0%	11** x2.2	9 (-)	-	-	6 -33%
Specify gravity	1040 (1010-1050)	1040 0%	1038 0%	1027* -1%	1035 (-)	-	-	1032 0%
pH	6.3 (6.0-7.0)	6.1 -3%	6.3 0%	6.3 0%	7.0 (-)	-	-	6.8 -3%

Statistically significant: *: p<0.05 and **: p<0.01.

In brackets, minimum-maximum values vs. Historical Control Data (HCD).

In italic: % vs. controls.

-: not applicable.

	Urinary parameters
Sex	Male
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
	End of treatment period				End of treatment-free period
Presence of proteins
. negative	0	1	2	10	1	-	-	3
. 0.3 g/L	2	5	8	0	2	-	-	2
. 1 g/L	7	4	0	0	2	-	-	0
.= 3 g/L	1	0	0	0	0	-	-	0
Presence of ketones
. negative	5	4	4	1	0	-	-	2
. 1.5 mmol/L	5	6	6	5	2	-	-	3
. 3.9 mmol/L	0	0	0	3	3	-	-	0
.= 7.8 mmol/L	0	0	0	1	0	-	-	0
Presence of bilirubin
. negative	2	5	7	10	5	-	-	5
. low	6	5	3	0	0	-	-	0
. moderate	2	0	0	0	0	-	-	0
. high	0	0	0	0	0	-	-	0

-: not applicable.

(continued)

		Urinary parameters
Sex		Male
Dose level (mg/kg/day)		0	100	300	1000	0		100	300	1000
	End of treatment period						End of treatment-free period
Presence of leucocytes
. none observed		8	7	8	4	1		-	-	2
. few in some fields		2	2	2	5	4		-	-	3
. few in all fields		0	1	0	1	0		-	-	0
. several in all fields		0	0	0	0	0		-	-	0
. large numbers in all fields		0	0	0	0	0		-	-	0
Presence of magnesium ammonium phosphate crystals
. none observed		0	1	0	8	1		-	-	0
. few in some fields		0	1	4	1	0		-	-	1
. few in all fields		9	5	6	1	2		-	-	3
. several in all fields		1	3	0	0	2		-	-	1
. large numbers in all fields		0	0	0	0	0		-	-	0
Presence of calcium oxalate crystals
. none observed		10	10	9	1	5		-	-	5
. few in some fields		0	0	0	1	0		-	-	0
. few in all fields		0	0	1	5	0		-	-	0
. several in all fields		0	0	0	3	0		-	-	0
. large numbers in all fields		0	0	0	0	0		-	-	0

-: not applicable.

 

		Urinary parameters
Sex		Female
Dose level (mg/kg/day)		0	100	300	1000	0		100	300	1000
	End of treatment period						End of treatment-free period
Presence of proteins
. negative		3	3	10	9	2		-	-	2
. 0.3 g/L		4	5	0	1	2		-	-	3
. 1 g/L		2	2	0	0	0		-	-	0
.= 3 g/L		1	0	0	0	0		-	-	0
Presence of ketones
. negative		10	10	10	10	4		-	-	5
. 1.5 mmol/L		0	0	0	0	0		-	-	0
. 3.9 mmol/L		0	0	0	0	0		-	-	0
.= 7.8 mmol/L		0	0	0	0	0		-	-	0
Presence of bilirubin
. negative		5	4	2	9	2		-	-	5
. low		1	2	6	1	2		-	-	0
. moderate		4	4	1	0	0		-	-	0
. high		0	0	1	0	0		-	-	0

-: not applicable.

(continued)

		Urinary parameters
Sex		Female
Dose level (mg/kg/day)		0	100	300	1000		0	100	300	1000
	End of treatment period					End of treatment-free period
Presence of leucocytes
. none observed		10	7	8	1		2	-	-	2
. few in some fields		0	0	1	3		2	-	-	3
. few in all fields		0	3	1	4		0	-	-	0
. several in all fields		0	0	0	2		0	-	-	0
. large numbers in all fields		0	0	0	0		0	-	-	0
Presence of magnesium ammonium phosphate crystals
. none observed		3	5	7	7		2	-	-	3
. few in some fields		6	2	2	3		2	-	-	2
. few in all fields		1	3	1	0		0	-	-	0
. several in all fields		0	0	0	0		0	-	-	0
. large numbers in all fields		0	0	0	0		0	-	-	0
Presence of calcium oxalate crystals
. none observed		8	7	3	1		4	-	-	5
. few in some fields		1	0	0	0		0	-	-	0
. few in all fields		1	3	6	2		0	-	-	0
. several in all fields		0	0	1	7		0	-	-	0
. large numbers in all fields		0	0	0	0		0	-	-	0

-: not applicable.

Table 11: Determination of serum levelsof anti-KLH IgM antibodies

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Predose	45.3	44.4	54.0	45.9	55.8	53.1	61.6	50.7
	-	-2	+19	+1	-	-5	+10	-9
Day 84	190	240	320	393	300	401	669	379
	-	+26	+68	+107	-	+34	+123	+26
Day 91	526	639	885	547	763	759	773	629
	-	+21	+68	+4	-	-1	+1	-18

Statistical analysis: no significance

In italic: % vs. controls.

 

Table 12: Determination of serum levels of anti-KLH IgG antibodies

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Predose	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	-	na	na	na	-	na	na	na
Day 84	0.0	11.7	7.0	25.3	13.0	18.8	63.1	23.2
	-	na	na	na	-	+45	+385	+78
Day 91	167	193	232	249	282	499	488	307
	-	+16	+39	+49	-	+77	+73	+9

Statistical analysis: no significance

In italic: % vs. controls.

na: not applicable; BLQ: < 20.0 µg/mL corresponding to 0.0 µg/mL for mean values

 

Table 13: Immunophenotyping

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000		0	100	300	1000
End of treatment period (principal animals)
B cells (cells/µL of blood)	3464.0	2913.5	2612.9*	2244.2**		2315.4	2100.3	2025.6	1834.1
	-	-15.9	-24.6	-35.2		-	-9.3	-12.5	-20.8
B cells (%)	41.1	39.4	36.4	33.5*		37.9	32.9	32.6	31.6
	-	-4.1	-11.4	-18.5		-	-13.2	-14.0	-16.6
Cytotoxic T cells (cells/µL of blood)	1192.6	1066.0	1195.3	1075.2		907.0	1103.1	928.3	1018.6
	-	-10.6	0.0	-9.8		-	+21.6	+2.3	+12.3
Cytotoxic T cells (%)	31.5	30.4	32.8	31.2		29.7	31.2	27.8	31.5
	-	-3.5	+4.1	-1.0		-	+5.1	-6.4	+6.1
End of treatment-free period (recovery animals)
B cells (cells/µL of blood)	3076	-	-	3702		1952	-	-	1683
	-	-	-	+20.4		-	-	-	-13.8
B cells (%)	38.6	-	-	45.4		33.9	-	-	29.9
	-	-	-	+17.6		-	-	-	-11.8
Cytotoxic T cells (cells/µL of blood)	1304	-	-	1037*		1180	-	-	1180
	-	-	-	-20.5		-	-	-	0.0
Cytotoxic T cells (%)	33.4	-	-	30.1		36.4	-	-	34.5
	-	-	-	-9.9		-	-	-	-5.2

In italic: % vs. controls.

Statistically significant *: p<0.05 and **: p<0.01

-: not applicable.

 

Table 14: Seminology

Dose level (mg/kg/day)	0	100	300	1000
End of treatment period (principal animals)
% of motile epididymal sperm	99.0	98.1	96.8	98.6
% of morphologically normal epididymal sperm	97.0	93.1	95.0	95.0
Mean number of epididymal sperm (10^6/cauda) % from controls	133.8 /	132.0 -1	139.8 +4	140.3 +5
Mean number of epididymal sperm (10^6/g cauda) % from controls	419.0 /	392.0 -6	416.7 -1	422.0 +1
Mean number of testicular sperm heads (10^6/g testis) % from controls	130.1 /	107.2* -18	107.8* -17	104.9* -19
Daily sperm production rate (10^6/g testis/day) % from controls	21.3 /	17.6* -17	17.7* -17	17.2* -19
End of treatment-free period (recovery animals)
Mean number of testicular sperm heads (106/g testis) % from controls	90.1 /	/ /	/ /	87.8 -3
Daily sperm production rate (106/g testis/day) % from controls	14.8 /	/ /	/ /	14.4 -3

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

 

Table 15: Organ weights

Selected differences in organ weights (% from controls)
Gender	Males			Females
Group	2	3	4	2	3	4
Dose (mg/kg/day)	100	300	1000	100	300	1000
Concentration (mg/mL)	20	60	200	20	60	200
Number of animals	10M	10M	10M	10F	10F	10F
Final body weight	-2	-7	-14**	+3	-3	-1
Adrenal glands
Absolute (%)	+2	-2	+2	-9	-5	+2
Relative to body weight (%)	+3	+5	+19*	-12	-2	+3
Kidneys
Absolute (%)	+4	+4	+10*	-4	-1	+18
Relative to body weight (%)	+5	+12	+28**	-6	+3	+19#
Liver
Absolute (%)	+9	+14*	+26**	+23	+29##	+51##
Relative to body weight (%)	+10**	+22**	+47**	+20**	+34**	+53**
Thymus
Absolute (%)	-13	-10	-37**	-3	-12	-23*
Relative to body weight (%)	-10	-2	-26*	-5	-7	-22#
Thyroid glands
Absolute (%)	-1	+1	-1	-1	-6	-9
Relative to body weight (%)	+1	+9	+15	-4	-3	-8

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

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

 

Table 16 : Microscopic examination - at the end of the treatment period

 

Incidence and severity of selected microscopic findings
Gender	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/kg)	0	100	300	1000	0	100	300	1000
Concentration (mg/mL)	0	20	60	200	0	20	60	200
Number of animals	10M	10M	10M	10M	10F	10F	10F	10F
Ileum
Dilatation, lateals
Minimal	0	0	0	4	0	0	1	3
Slight	0	0	0	5	0	0	0	1
Kidneys
Tubular basophilia
Minimal	0	6	6	2	0	0	0	1
Slight	0	1	3	3	0	0	0	0
Moderate	0	0	0	2	0	0	0	0
Cast, granular
Minimal	0	2	2	2	0	0	0	0
Slight	0	0	3	3	0	0	0	0
Moderate	0	0	0	3	0	0	0	0
Pyelitis
Slight	0	0	0	0	0	3	0	0
Moderate	0	0	0	0	0	0	0	1
Increased hyaline droplets
Minimal	0	6	4	4	0	0	0	0
Slight	0	0	3	4	0	0	0	0
Moderate	0	0	1	0	0	0	0	0
Mineralization, cortico-medullary junction
Present	0	0	0	7	2	0	0	2
Pyelonephritis
Moderate	0	0	0	0	0	1	0	2
Positive for alpha 2u globulin IHC
Minimal	6	-	-	2	-	-	-	-
Slight	4	-	-	4	-	-	-	-
Moderate	0	-	-	4	-	-	-	-

 

Incidence and severity of selected microscopic findings
Gender	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/kg)	0	100	300	1000	0	100	300	1000
Concentration (mg/mL)	0	20	60	200	0	20	60	200
Number of animals	10M	10M	10M	10M	10F	10F	10F	10F
Liver
Centrilobular hypertrophy
Minimal	0	0	0	5	0	0	5	6
Slight	0	0	0	4	0	0	1	4
Vacuolation, Kupffer cells
Minimal	0	0	0	9	0	0	0	8
Slight	0	0	0	0	0	0	0	1
Thyroid glands
Follicular cell hypertrophy
Minimal	0	0	0	7	0	0	0	7
Thymus
Lymphoid atrophy, cortex
Minimal	0	0	0	5	1	0	0	5

 

Table 17: Microscopic examination - at the end of the recovery period

Incidence and severity of selected microscopic findings (compared to end of treatment)
Gender	Males				Females
Group	1	1	4	4	1	1	4	4
		recovery		recovery		recovery		recovery
Dose (mg/kg/day)	0	0	1000	1000	0	0	1000	1000
Concentration (mg/mL)	0	0	200	200	0	0	200	200
Number of animals	10	5	10	5	10	5	10	5
Ileum
Dilatation, lacteals
Minimal	0	0	4	0	0	0	3	1
Slight	0	0	5	0	0	0	1	0
Kidney
Tubular basophilia
Minimal	0	0	2	3	0	0	1	0
Slight	0	0	3	1	0	0	0	0
Moderate	0	0	2	0	0	0	0	0
Cast, granular
Minimal	0	0	2	0	0	0	0	0
Slight	0	0	3	0	0	0	0	0
Moderate	0	0	3	0	0	0	0	0
Pyelitis
Slight	0	0	0	0	0	0	0	1
Moderate	0	0	0	0	0	0	1	0
Increased hyaline droplets
Minimal	0	6	4	0	0	0	0	0
Slight	0	0	4	0	0	0	0	0
Moderate	0	0	0	0	0	0	0	0
Mineralization, cortico-medullary junction
Present	0	0	7	0	2	0	2	0
Pyelonephritis
Moderate	0	0	0	0	0	0	2	0

 

Incidence and severity of selected microscopic findings (compared to end of treatment)
Gender	Males				Females
Group	1	1	4	4	1	1	4	4
		recovery		recovery		recovery		recovery
Dose (mg/kg/day)	0	0	1000	1000	0	0	1000	1000
Concentration (mg/mL)	0	0	200	200	0	0	200	200
Number of animals	10	5	10	5	10	5	10	5
Liver
Centrilobular hypertrophy
Minimal	0	0	5	0	0	0	6	0
Slight	0	0	4	0	0	0	4	0
Vacuolation, Kupffer cells
Minimal	0	0	9	0	0	0	8	0
Slight	0	0	0	0	0	0	1	0
Thyroid glands
Follicular cell hypertrophy
Minimal	0	0	7	0	0	0	7	0
Thymus
Lymphoid atrophy, cortex
Minimal	0	0	5	1	1	0	5	0

 

Applicant's summary and conclusion

Conclusions:

The toxicity of the test item was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day for 13 weeks followed by a 6-week treatment-free period.
Under the experimental conditions of the study, adverse test item related effects consisted of a2u-globulin nephropathy in males from the dose level of 300 mg/kg/day.

Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 100 mg/kg/day in males and 1000 mg/kg/day in females. However, the nature of the histopathological changes in the kidneys of males (a2u-globulin nephropathy) are considered to be of no toxicological relevance for human risk assessment.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item, 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).

 

Methods

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 1000 mg/kg/day (group 4) for 13 weeks. Two other groups of 10 males and 10 females were treated with the test item at the dose level of 100 or 300 mg/kg/day (groups 2 and 3, respectively). One control group of 15 males and 15 females received the vehicle only (sesame oil) under the same experimental conditions (group 1). A constant dosage volume of 5 mL/kg/day was used.

At the end of the treatment period, the animals were euthanized, except for the first five group 1 and group 4 animals per sex, which were kept for a 6-week treatment-free period.

Eight satellite animals per sex were included in each group for T-cell Dependant Antibody Response (TDAR) evaluation only.

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 8 and 13 were determined using a High Performance Liquid Chromatography with UV detection method (HPLC/UV).

 

The animals were checked at least once daily for mortality and clinical signs. Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 12.

Body weight was recorded pre-test, on the first day of treatment and then once a week. Food and water consumption was recorded weekly.

Ophthalmological examinations were performed on all animals before the beginning of the treatment period and on control and high-dose animals euthanized at the end of the treatment period (Week 13).

On Days 77 (Week 11) and 84 (Week 12), satellite animals were immunized with KLH in order to evaluate the immune response. For this purpose, blood was taken in pre-dose and on Days 84 (Week 12) and 91 (Week 13) for determination of specific anti-KLH IgM and IgG levels.

Hematology, blood biochemistry, urinary and immunophenotyping investigations were performed on all animals euthanized at the end of the treatment period (Week 13). All these investigations were also performed for all animals euthanized at the end of treatment-free period (Week 20). Additional blood samples were collected in Weeks 13 and 20 for the analysis of T4, T3 and TSH thyroid hormone levels in all animals euthanized at the end of the treatment and treatment-free periods.

The estrus cycle was determined over 21 consecutive days for all principal females at the end of the treatment period. At the end of the treatment period, seminological investigations (sperm count, motility and morphology) were performed for all males. Testicular sperm count investigations were also carried out in control and high-dose males at the end of the treatment-free period.

On completion of the treatment or treatment-free period, the animals were euthanized and a full macroscopic post-mortem examination was performed (with the exception of satellite animals which were discarded without any further investigations). Designated organs were weighed and selected tissues were preserved. A microscopic examination (including a detailed examination of the testes) was performed on designated tissues from control and high-dose animals euthanized at the end of the treatment period and from animals that were euthanized prematurely, and on all macroscopic lesions from low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period.

A microscopic examination was also performed on kidney slides (immunostained with an antibody for a2u-globulin protein) from all control and high-dose males euthanized at the end of the treatment period. The ileum, kidneys, liver, thyroids with parathyroids, and thymus were also microscopically examined for low- and intermediate-dose animals (groups 2 and 3) euthanized at the end of the treatment period, and for the recovery animals (groups 1 and 4) as changes were noted in these organs at the end of the treatment period.

 

Results

Actual concentrations of the test item in the dose formulations administered to the animals during the study remained within an acceptable range (-0.3% to +8.0%) compared to the nominal concentrations.

 

There were no test item-related unscheduled deaths in any group.

 

Ptyalism was observed in control males and in males and females at all dose levels with a dose-related incidence. This sign was considered to be non-adverse.

 

The FOB results showed slight differences of horizontal and rearing movements without toxicological importance.

 

Lower body weight gain was recorded throughout the treatment period in males given 300 or 1000 mg/kg/day (-14% and -20% vs.controls, respectively), leading to minimally lower body weights on completion of the treatment period (-7% and -13% vs. controls, respectively). As these differences were of low magnitude and/or reversible at the end of the treatment-free period, they were considered to be non-adverse. Body weight change of females given 300 or 1000 mg/kg/day fluctuated throughout the treatment period with no relevant effects on the body weight.

 

Food consumption was slightly to moderately lower (9% to -13%) in males given 1000 mg/kg/day than in controls for five weeks at the beginning of the treatment period, and the same trend was noted in males given 300 mg/kg/day in Week 4 (-9%). As these differences were transient and/or reversible, they were considered to be non-adverse although they partially correspond to the lower mean body weight gain recorded in males at these dose levels. Food consumption of females was not affected by the test item.

 

Water consumption was higher throughout the treatment period in males (+13% to +57% from Week 2 to Week 13 vs. controls) and females (+29 to +99% from Week 1 to Week 13 vs. controls) given 1000 mg/kg/day. These moderate to marked differences increased over time during the treatment period and were still observed during the treatment-free period but to a lesser extent, suggesting partial recovery. However, these changes were not considered as adverse but rather adaptive changes as they did not correlate with any adverse findings. Minor, sporadic and non-adverse higher water intake was also noted in males given 300 mg/kg/day.

 

No test item-related ophthalmological findings were observed at the end of the treatment period.

Estrus cycles were unaffected by the test item.

The epididymal sperm motility, morphology and count were unaffected by the test item. Statistically significant, lower testicular sperm counts were recorded in males from 100 mg/kg/day (-17% to -19% vs. controls). In view of their magnitude, in the absence of a dose relationship and absence of microscopic correlates, and as the effects were reversible, these differences were considered to be non-adverse.

At hematology investigations and when compared with controls, minimal decreases in red blood cell parameters were noted in males and/or females given 1000 mg/kg/day, including red blood cell count (males only: -7%), hemoglobin concentration (males: -5% and females: -6%) and packed cell volume (males: -6% and females: -4%). These changes were noted along with slightly lower reticulocyte count in males (-19%) and females (-16%). At 1000 mg/kg/day, slightly decreased white blood cell count was recorded in males (-18%) in association with lower eosinophil count (-45%) and lower lymphocyte count (-20%). At 1000 mg/kg/day, shortened prothrombin time was also recorded in males (-8%) and to a lesser extent in females (-5%).

All hematological effects were considered to beof minor toxicological importance in view of their magnitude and reversibility (return to the range of control values), as values remained within the range of the Historical Control Data and/or as they correlated with non-adverse histopathological findings and/or with non-adverse decreased B cell population subset.

 

At blood biochemistry investigations and when compared with controls, the following changes were noted and consisted of:

. lower potassium level in females at 100, 300 and 1000 mg/kg/day (-6% to -8%) and males at 1000 mg/kg/day (-5%),

. higher inorganic phosphorus level in females at 1000 mg/kg/day (+9%),

. higher urea level in males at 300 mg/kg/day (+15%) and in males and females at 1000 mg/kg/day (+18% and +34%, respectively) associated with higher creatinine level in males and females at 1000 mg/kg/day (+9% and +15%, respectively),

. higher protein level in females at 100, 300 and 1000 mg/kg/day (+11%, +9% and +10%, respectively) and in males at 1000 mg/kg/day (+3%), together with higher calcium level in males and females at 300 mg/kg/day (+3% and +5%, respectively) and 1000 mg/kg/day (+6% and +13%, respectively),

. higher albumin level in females at all dose levels (+8%) and lower albumin/globulin ratio level in males at 1000 mg/kg/day (-5%),

. minimal decrease in glucose level in males and females at 1000 mg/kg/day (-10%).

In addition, increased cholesterol levels were recorded in males and females at 100 (+42% and +47%, respectively), 300 (+55% and +88%, respectively) and 1000 (2.5- and 2.0-fold, respectively) mg/kg/day, as well as a minimal increase of alkaline phosphatase activity in males given 1000 mg/kg/day (+34%). They were considered to be most probably related to hepatocellular hypertrophy.

All these changes in biochemistry parameters were no longer observed at the end of the treatment-free period with the exception of urea level (+19% vs. controls) in males previously given 1000 mg/kg/day, which remained increased but closed to the upper limit of the Historical Control Data. All blood biochemical effects at the end of the treatment or treatment-free period were considered to be of minor toxicological importance in view of their magnitude and/or reversibility, as values remained within the range of the Historical Control Data and/or did not correlate with histopathological findings.

 

At urinary investigations and when compared with controls, all changes observed at 1000 mg/kg/day namely higher urinary volumes in males and females (2-fold and 2.2-fold, respectively), along with slightly decreased specific gravity in females (-1%) or lower pH in males (-13%), presence of ketones (= 3.9 mmol/L) in males, and presence of calcium oxalate crystals in males and females were reversible and considered to be of minor toxicological importance. Some of these differences (i.e. calcium oxalate crystals) were also noted in females given 300 mg/kg/day, but to a lesser extent.

 

At T-Cell Dependant Antibody Response evaluation, when compared with control groups, no statistically significant or meaningful decrease/increase in anti-KLH IgM and IgG concentrations in rat serum were observed in test item-treated groups. Indeed, there is no evidence of toxic effects on the immune system.

At immunology investigations and when compared with controls, B lymphocyte subset was found to be decreased in males given 300 or 1000 mg/kg/day in terms of both relative (-18.5%) and absolute count (-24.6% and -35.2% at 300 and 1000 mg/kg/day, respectively). Same decrease was observed in females given 1000 mg/kg/day without statistically significance (absolute and relative counts -20.8% and -16.6%, respectively). These variations were dose-related in males and females and were statistically significant in males. They were considered to be non adverse in view of their magnitude, of the absence of functional correlates at TDAR evaluation, and of their correlation with non-adverse lymphoid atrophy (fully reversible) and/or their reversibility. No test item-related changes in T lymphocyte subset were observed at any dose levels.

 

At thyroid hormones investigations and when compared with controls, T3 and TSH levels were increased in males (+32% and +200%, respectively) and in females (+22% and +197%, respectively) and T4 concentration was higher in females (+33%) at 1000 mg/kg/day. These results correlated with thyroid follicular cell hypertrophy at 1000 mg/kg/day in a context of hepatic centrilobular hypertrophy. At the end of the treatment-free period, slightly higher T3 levels (29% vs. controls) were still observed in females indicating on-going recovery. These effects correlated with non-adverse thyroid cell hypertrophy at microscopy and were considered to be an indirect effect of the test item through the non-adverse increase in hepatic metabolism. They were therefore considered to be non-adverse.

 

At pathology investigations, no macroscopic change was observed. However, a2u-globulin nephropathy was observed in male rats at all dose levels (as confirmed by immunohistochemistry). This finding was considered to be adverse from the dose of 300 mg/kg, but it is irrelevant for human risk assessment.

In addition, there was also Kupffer cell vacuolation in the liver at 1000 mg/kg/day in both sexes, hepatic centrilobular hypertrophy (from 300 mg/kg/day in females, and in males given 1000 mg/kg/day) accompanied by thyroid follicular cell hypertrophy at 1000 mg/kg/day in both sexes, dilatation of lacteals (i.e. lymphangiectasis) in the ileum (from 300 mg/kg/day in females and in males given 1000 mg/kg/day) and thymic cortical atrophy at 1000 mg/kg/day in both sexes, which were all considered as non-adverse.

At the end of the 6-week treatment-free period, none of these changes were observed, thus indicating full recovery, with the exception of basophilic tubules in the kidney (in males) and dilatation of lacteals in the ileum (in one female), for which there was evidence of ongoing recovery.

The qualitative assessment of the bone marrow did not show test item-related findings.

 

Conclusion

The toxicity of the test item was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day for 13 weeks followed by a 6-week treatment-free period.

 

Under the experimental conditions of the study, adverse test item-related effects consisted of a2u-globulin nephropathy in males from the dose level of 300 mg/kg/day.

 

Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 100 mg/kg/day in males and 1000 mg/kg/day in females. However, the nature of the histopathological changes in the kidneys of males (a2u-globulin nephropathy) are considered to be of no toxicological relevance for human risk assessment