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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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

Data source

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

Materials and methods

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

Test material

1
Reference substance name:
A mixture of: triphenylthiophosphate and tertiary butylated phenyl derivatives
EC Number:
421-820-9
EC Name:
A mixture of: triphenylthiophosphate and tertiary butylated phenyl derivatives
Cas Number:
192268-65-8
Molecular formula:
Unspecified
IUPAC Name:
A mixture of: triphenylthiophosphate and tertiary butylated phenyl derivatives

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 34 ± 1 days
- Weight at study initiation:
- Housing: together (5 animals per cage) in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2)
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum, from water bottles
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, polyethylene glycol (PEG 400) was filled up to the desired volume and subsequently homogenized with a magnetic stirrer. Additionally, the preparations were treated in an ultrasonic bath for at least 5 minutes. The test-substance preparations were produced at least once a week and stored at room temperature. The administration volume was 4 mL/kg body weight.

VEHICLE
- Concentration in vehicle: 0.25, 1.25, 6.25 g/100 ml
- Amount of vehicle (if gavage): 4 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in polyethylene glycol (PEG 400) at room temperature for a period of 7 days was proven. At the beginning of the administration period samples were taken from the lowest and highest concentration for homogeneity analysis. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test item was distributed homogeneously in polyethylene glycol. Additional concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start of the administration period. Moreover, concentration control analyses were verified in 1 sample of each concentration towards the end of the administration period. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90-110% of the nominal concentrations. Taken together, the results demonstrate the correctness of the concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 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)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: Abnormal behavior when handled, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Impairment of gait, Lacrimation, Palpebral closure, Exophthalmus, Feces (appearance/ consistency), Urine, Pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period, the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume until study day 32. Since an increased water consumption was monitored, water consumption was determined once a week from study day 33 onwards.

OPHTHALMOSCOPIC EXAMINATION: Yes
Prior to the start of the administration period on day -1 the eyes of all animals and on study day 90 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope after administration of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes. Blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. Clotting tests (Prothrombin time) were carried out using a ball coagulometer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, γ-Glutamyltransferase, Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: during the night
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Home cage observations: The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the rats. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Other findings
- Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/ pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypes, Gait abnormalities, Activity/ arousal level, Feces excreted within 2 minutes (appearance/ consistency), Urine excreted within 2 minutes (amount/ color), Rearing within 2 minutes, Other findings
- Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (approach response), Touch sensitivity (touch response), Vision (visual placing response), Pupillary reflex, Pinna reflex, Audition (auditory startle response), Coordination of movements (righting response), Behavior during handling, Vocalization, Pain perception (tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test, Other findings
- Motor activity assessment: Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the rats during these measurements and the measurement room was darkened after the transfer of the last rat.

OTHER:
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were euthanized one day after the end of the treatment period (males on study day 92 and females on study day 93). The euthanized animals were necropsied and subjected to a full macroscopic evaluation. The animals were sacrificed by decapitation under isoflurane anesthesia.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix

HISTOPATHOLOGY: Yes
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution. The left testis and left epididymis of all animals were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters. In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm parameters. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below.

SPERM PARAMETERS
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male on schedule: Sperm motility, Sperm morphology, Sperm head count (cauda epididymis),
Sperm head count (testis). Sperm motility examinations were carried out in a randomized sequence. Sperm head count (testis) were determined in the control and highest dose group, only.
Statistics:
- Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
- Rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity, estrous cycle, Blood parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians
- Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. In case of exactly the same numbers of the dose group and the control, no statistical test is performed.
- Urine pH, volume, specific gravity and Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians.
- Sperm analysis parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians; If only control and one dose group are measured, WILCOXON-test (one-sided) without adjustment were used. For the percentage of abnormal sperms (ABNORMAL6_C) values < 6% were set to 6% (cut off 6%)

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related, adverse effects were obtained in any test group (10, 50 and 250 mg/kg bw/d).
Respiratory sounds were observed in 2 male animals (No. 31 from study day 76 until study day 87 and No. 36 on study day 91) of test group 3 (250 mg/kg bw/d). The findings were considered to be related to the administration method as such, as it was considered that these animals aspirated parts of the test substance preparation. A specific systemic effect of the test substance on the respiration tract was excluded, also taking into account that no potentially related findings occurred during pathological examinations.
Salivation after treatment was observed in all male and all female animals of test groups 3 (250 mg/kg bw/d) and 2 (50 mg/kg bw/d) on several days of the study. From the temporary, short appearance immediately after dosing it was concluded that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant treatment-related changes for body weight parameters occurred in male and female animals of test groups 1-3 (10, 50 and 250 mg/kg bw/d) when compared to the control animals of test group 0.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to food consumption were observed.
Note: On study days 77 and 84 food spilling was observed in cage No. 7 of male animals of test group 3 (250 mg/kg bw/d). Thus, these values were excluded from the calculation of mean values.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
During daily visual inspection of the water bottles for any changes in volume, drinking water consumption seemed to be increased during the first third of the administration period. Since then, water consumption was determined once a week (from study day 33 onwards). However, no test substance-related, adverse changes with regard to water consumption were observed.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were observed.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in males of test group 3 (250 mg/kg bw/d) prothrombin time (i.e., Hepatoquick’s test, HQT) was significantly prolonged. In males of the mentioned test group, absolute and relative monocyte counts were significantly decreased. Both mean values were below historical control ranges (males, absolute monocytes 0.08-0.15 Giga/L; relative monocytes 1.6-2.8 %). An isolated monocytopenia is very seldom. No other differential blood cell count was altered in these individuals nor in the corresponding sex. Therefore, the lower monocyte counts were regarded as maybe treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in males of test group 3 (250 mg/kg bw/d) cholesterol levels were significantly decreased, and in females of the same test group γ-glutamyl transferase (GGT) activities and inorganic phosphate levels were significantly increased. Inorganic phosphate values were already higher in females of test group 2 (50 mg/kg bw/d), but in this group, it was the only changed clinical pathology parameter. Therefore, at the intermediate dose level this alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
In male and female rats of test group 3 (250 mg/kg bw/d) and in males of test group 2 (50 mg/kg bw/d) total bilirubin levels were significantly decreased. Lower serum bilirubin values were due to a higher conjugation rate by induced liver cells, followed by an accelerated excretion of bilirubin via the bile. This mechanism was regarded as treatment-related, but not adverse.
In female rats of test groups 2 and 3 (50 and 250 mg/kg bw/d) alanine aminotransferase (ALT) activities were significantly higher compared to controls. The ALT mean value in test group 3 was 1.5-fold higher compared to that of the controls. This increase was regarded as treatment-related, but not adverse (Hall et al., 2012).
In females of test group 3 (250 mg/kg bw/d) serum chloride levels were significantly decreased. However, only this electrolyte level was changed without any alteration of sodium and potassium levels and without any change of electrolyte levels in males. Therefore, chloride decrease in females of test group 3 was regarded as treatment-related, but not adverse.
The following clinical chemistry parameter changes were within historical control ranges and, therefore, they were regarded as incidental and not treatment-related: alkaline phosphatase (ALP) activity and albumin level increase in males of test group 3; total protein, globulin and calcium levels changes in females of test groups 1, 2 and 3 (males, ALP 0.97-1.35 µkat/L; albumin 33.28-40.67 g/L; females, total protein 63.21-70.49 g/L; globulins 21.63-32.75 g/L; calcium 2.50-2.69 mmol/L).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In males of test group 3 (250 mg/kg bw/d) incidences of urine triple phosphate crystals and epithelial and granular casts were increased.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Deviation from "zero values" was obtained in one male animal of test group 3 (250 mg/kg bw/d). However, since this finding was without a dose-response relationship and occurred in a single animal only, this observation was considered to have been incidental.
The following examinations were performed during FOB and have to be assessed individually:
- Home cage observations: No test substance-related effects were observed. Respiratory sounds occurred in male animal No. 31 of test group 3 (250 mg/kg bw/d) but the change was not considered to be related to a systemically toxic effect of the test substance.
- Open field observations: No test substance-related effects were observed. Respiratory sounds occurred in male animal No. 31 of test group 3 (250 mg/kg bw/d) but the change was not considered to be related to a systemically toxic effect of the test substance.
- Sensorimotor tests/reflexes: No test substance-related effects were observed. Respiratory sounds occurred in male animal No. 31 of test group 3 (250 mg/kg bw/d) but the change was not considered to be related to a systemically toxic effect of the test substance.
- Quantitative parameters: No test substance-related effects were observed.
- Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for male and female animals of test groups 1-3 (10, 50 and 250 mg/kg bw/d).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Possible test item-related organ weight changes are:
- Liver: Mean absolute and relative liver weights were dose-relatedly statistically significantly increased in males and females of the 50 and 250 mg/kg bw/d treated group.
- Adrenal glands: A slight, statistically significant increase of the mean absolute and relative adrenal gland weights were present in females treated with 250 mg/kg bw/d.
- Ovaries: A slight, statistical significant increase of the mean absolute and relative ovarian weight was present in females treated with 250 mg/kg bw/d.
- Thyroid glands: Minimal increased mean absolute and relative thyroid gland weights were present in males treated with 250 mg/kg bw/d.
All other organ weight and/or organ weight ratio changes even when statistically significantly deviating from controls (such as the absolute mean epididymal weight in group 2 and 3 males, relative mean kidney weight in males of group 3, absolute thyroid gland weight in females of group 1, and absolute and relative mean uterine weight in females of group 1) were considered unrelated to treatment based on small differences in mean terminal body weight, absence of a dose response and/or lack of correlating microscopic findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A (dark brown) discolored liver was observed in 7/10 males and 8/10 females from test group 3 treated with 250 mg/kg bw/d. All other macroscopic findings reported in the individual animal table are considered spontaneous and consistent with the usual pattern of findings in animals of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test-item related histopathologic findings:
Liver:
- Centrilobular hepatocellular hypertrophy was observed in 7/10 females of the 50 mg/kg bw/d and in all males and females treated with 250 mg/kg bw/d.In females the incidences and severity of the hypertrophy showed a clear dose-relationship.
- Intrahepatocellular (fine brown) pigment accumulation was observed in 4/10 males and 8/10 females treated with 250 mg/kg bw/d. Using the Schmorl’s, PAS, Hall and Perls’s method the intrahepatocellular pigment was proven to consist of lipfuscin mainly.
- (Per)vasculitis associated with the branches of the hepatic artery was noted in a single male and female of the 50 mg/kg bw/d treated group, and in 3/10 males of test group 3 treated with 250 mg/kg bw/d. In most affected animals, the lesion was characterized by degeneration and/or fibrinoid necrosis and perivasculitis. In one other female of the 250 mg/kg bw/d treated group the lesion only consisted of degeneration/necrosis of the arterial wall of a branch of the hepatic artery. In the affected male of the 50 mg/kg bw/d treated group, the vasculitis could be related to an area of hepatocellular necrosis.
Ovaries:
- Minimal increased interstitial cell vacuolation was observed in 6 out of 10 females of the group treated with 250 mg/kg bw/d.

Uncertain test-item related histopathologic findings:
Kidney:
- Slight increased severity of hyaline droplet accumulation, as determined by the CAB staining method was noted in males of test group 3 treated with 250 mg/kg bw/d. In few selected control and high-dose group animals, the hyaline droplets were proven to mainly consist of alpha2U-globulin using an anti-body raised against this protein.
Thyroid gland:
- Minimal follicular cell hypertrophy was observed in 1/10 males treated with 250 mg/kg b/d.
- Minimal colloid alteration (inspissated colloid) was noted in 3/10 males and a single female treated with 250 mg/kg bw/d and in a single control male.

Other microscopic findings reported in the individual animal table are considered spontaneous or incidental and consistent with the usual pattern of findings in animals of this strain and age.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No test substance-related effects on estrous cycle length and the number of cycles were obtained. Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis, no treatment-related effects were observed.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes

Any other information on results incl. tables

CLINICAL CHEMISTRY

Red Blood Cell + coagulation parameters

Males Dose Group 0 1 2 3
RBC Mean 8.36 k 8.22 8.43 8.18
[tera/L] S.d. 0.33 0.32 0.31 0.23
day 92 N 10 10 10 10
Median 8.35 8.28 8.54 8.11
  Deviation Vs Control   -1.65 0.87 -2.16
HGB Mean 8.9 k 8.7 8.9 8.7
[mmol/L] S.d. 0.3 0.1 0.2 0.2
day 92 N 10 10 10 10
Median 8.9 8.7 8.9 8.7
  Deviation Vs Control   -2.0 0.1 -2.4
HCT Mean 0.412 k 0.412 0.417 0.410
[L/L] S.d. 0.013 0.012 0.010 0.011
day 92 N 10 10 10 10
Median 0.411 0.413 0.418 0.407
  Deviation Vs Control   -0.097 1.286 -0.582
MCV Mean 49.4 k 50.1 49.5 50.1
[fL] S.d. 1.7 1.9 1.3 1.3
day 92 N 10 10 10 10
Median 49.5 49.8 49.0 50.2
  Deviation Vs Control   1.5 0.3 1.5
MCH Mean 1.07 k 1.06 1.06 1.07
[fmol] S.d. 0.04 0.04 0.04 0.03
day 92 N 10 10 10 10
Median 1.06 1.06 1.06 1.08
  Deviation Vs Control   -0.19 -0.47 0.00
MCHC Mean 21.61 k 21.26 21.41 21.29
[mmol/L] S.d. 0.39 0.49 0.32 0.43
day 92 N 10 10 10 10
Median 21.55 21.14 21.50 21.34
  Deviation Vs Control   -1.62 -0.91 -1.45
RETA Mean 141.5 k 137.1 135.8 123.2
[giga/L] S.d. 17.0 11.3 15.8 21.8
day 92 N 10 10 10 10
Median 144.6 138.3 141.5 121.4
  Deviation Vs Control   -3.1 -4.0 -12.9
PLT Mean 722 k 728 732 761
[giga/L] S.d. 61 70 110 91
day 92 N 9 10 10 10
Median 720 747 732 760
  Deviation Vs Control   1 1 5
HQT Mean 36.2 v 36.5 38.2 42.4 **
[sec] S.d. 1.6 2.2 2.3 4.5
day 92 N 10 10 10 10
Median 36.1 36.6 37.7 41.2
  Deviation Vs Control   0.9 5.4 17.0

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS; v=KRUSKAL-WALLIS-WILCOX

Females Dose Group 0 1 2 3
RBC Mean 7.35 k 7.36 7.41 7.42
[tera/L] S.d. 0.23 0.27 0.31 0.35
day 93 N 10 10 10 10
Median 7.37 7.34 7.39 7.54
  Deviation Vs Control   0.14 0.83 0.97
HGB Mean 8.3 k 8.4 8.3 8.2
[mmol/L] S.d. 0.2 0.2 0.2 0.2
day 93 N 10 10 10 10
Median 8.4 8.5 8.3 8.2
  Deviation Vs Control   1.0 -0.1 -1.1
HCT Mean 0.387 k 0.389 0.387 0.379
[L/L] S.d. 0.009 0.010 0.012 0.010
day 93 N 10 10 10 10
Median 0.386 0.392 0.387 0.379
  Deviation Vs Control   0.491 0.026 -2.092
MCV Mean 52.8 k 52.9 52.3 51.2
[fL] S.d. 1.8 1.3 1.2 1.8
day 93 N 10 10 10 10
Median 53.2 53.1 52.3 51.2
  Deviation Vs Control   0.3 -0.8 -3.0
MCH Mean 1.13 k 1.15 1.13 1.11
[fmol] S.d. 0.04 0.05 0.03 0.04
day 93 N 10 10 10 10
Median 1.15 1.16 1.13 1.11
  Deviation Vs Control   0.97 -0.70 -1.94
MCHC Mean 21.51 k 21.69 21.54 21.74
[mmol/L] S.d. 0.42 0.53 0.33 0.39
day 93 N 10 10 10 10
Median 21.39 21.76 21.54 21.68
  Deviation Vs Control   0.82 0.14 1.04
RETA Mean 143.6 k 141.8 157.1 164.8
[giga/L] S.d. 23.5 33.3 20.3 39.7
day 93 N 10 10 10 10
Median 136.6 147.7 161.2 167.1
  Deviation Vs Control   -1.2 9.4 14.8
PLT Mean 725 k 686 728 784
[giga/L] S.d. 89 44 86 104
day 93 N 10 10 10 10
Median 737 688 722 772
  Deviation Vs Control   -5 1 8
HQT Mean 35.1 k 33.7 34.3 32.6
[sec] S.d. 2.5 1.4 1.7 1.9
day 93 N 10 10 10 10
Median 35.6 33.9 34.2 32.7
  Deviation Vs Control   -4.0 -2.3 -7.0

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS

Total white and differential blood cell count

Males Dose group 0 1 2 3
WBC Mean 5.42 k 5.29 5.82 5.40
[giga/L] S.d. 1.19 0.98 0.99 1.44
day 92 N 10 10 10 10
Median 5.48 5.36 5.71 4.85
  Deviation Vs Control   -2.56 7.24 -0.37
NEUTA Mean 1.02 k 0.94 1.10 1.20
[giga/L] S.d. 0.15 0.13 0.19 0.35
day 92 N 10 10 10 10
Median 1.00 0.96 1.04 1.10
  Deviation Vs Control   -8.06 7.86 17.88
LYMPHA Mean 4.17 k 4.10 4.49 4.02
[giga/L] S.d. 1.10 0.94 0.86 1.08
day 92 N 10 10 10 10
Median 4.11 4.14 4.41 3.58
  Deviation Vs Control   -1.73 7.65 -3.53
MONOA Mean 0.11 v 0.12 0.10 0.07 *
[giga/L] S.d. 0.03 0.04 0.04 0.02
day 92 N 10 10 10 10
Median 0.11 0.11 0.09 0.06
  Deviation Vs Control   10.48 -4.76 -32.38
EOSA Mean 0.10 k 0.11 0.11 0.09
[giga/L] S.d. 0.03 0.04 0.04 0.05
day 92 N 10 10 10 10
Median 0.10 0.11 0.09 0.08
  Deviation Vs Control   8.65 0.96 -12.50
BASOA Mean 0.01 k 0.01 0.01 0.01
[giga/L] S.d. 0.01 0.01 0.00 0.00
day 92 N 10 10 10 10
Median 0.01 0.01 0.01 0.01
  Deviation Vs Control   0.00 18.18 -18.18
LUCA Mean 0.02 k 0.01 0.02 0.01
[giga/L] S.d. 0.01 0.01 0.01 0.01
day 92 N 10 10 10 10
Median 0.01 0.01 0.01 0.01
  Deviation Vs Control   -22.22 -16.67 -16.67
NEUT Mean 19.4 k 18.1 19.1 22.2
[%] S.d. 3.8 3.7 3.3 2.7
day 92 N 10 10 10 10
Median 19.6 17.0 17.9 22.3
  Deviation Vs Control   -6.6 -1.5 14.7
LYMPH Mean 76.2 k 77.0 76.9 74.3
[%] S.d. 4.0 4.3 3.7 3.1
day 92 N 10 10 10 10
Median 76.0 77.5 77.9 74.2
  Deviation Vs Control   1.0 0.9 -2.5
MONO Mean 1.9 v 2.2 1.7 1.4 **
[%] S.d. 0.6 0.6 0.4 0.3
day 92 N 10 10 10 10
Median 1.8 2.2 1.7 1.3
  Deviation Vs Control   12.9 -12.9 -29.4
EOS Mean 1.9 k 2.3 1.8 1.6
[%] S.d. 0.5 0.9 0.8 0.6
day 92 N 10 10 10 10
Median 1.9 2.0 1.6 1.5
  Deviation Vs Control   16.6 -5.2 -15.0
BASO Mean 0.2 k 0.3 0.2 0.1
[%] S.d. 0.1 0.1 0.1 0.1
day 92 N 10 10 10 10
Median 0.2 0.2 0.2 0.1
  Deviation Vs Control   13.6 -9.1 -36.4
LUC Mean 0.3 k 0.2 0.2 0.3
[%] S.d. 0.2 0.1 0.1 0.1
day 92 N 10 10 10 10
Median 0.2 0.2 0.2 0.3
  Deviation Vs Control   -17.2 -13.8 3.4

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS; v=KRUSKAL-WALLIS-WILCOX

Females Dose group 0 1 2 3
WBC Mean 3.01 k 3.44 3.57 4.00
[giga/L] S.d. 0.63 0.71 1.15 0.83
day 93 N 10 10 10 10
Median 2.95 3.47 3.12 3.91
  Deviation Vs Control   14.47 18.62 32.96
NEUTA Mean 0.51 k 0.57 0.66 0.62
[giga/L] S.d. 0.14 0.17 0.23 0.18
day 93 N 10 10 10 10
Median 0.53 0.55 0.61 0.61
  Deviation Vs Control   11.94 28.38 20.94
LYMPHA Mean 2.33 k 2.69 2.71 3.20
[giga/L] S.d. 0.61 0.57 0.95 0.69
day 93 N 10 10 10 10
Median 2.40 2.58 2.52 3.16
  Deviation Vs Control   15.62 16.31 37.30
MONOA Mean 0.06 k 0.07 0.08 0.08
[giga/L] S.d. 0.03 0.03 0.03 0.02
day 93 N 10 10 10 10
Median 0.06 0.07 0.07 0.08
  Deviation Vs Control   16.67 31.67 31.67
EOSA Mean 0.09 k 0.08 0.10 0.08
[giga/L] S.d. 0.05 0.03 0.05 0.03
day 93 N 10 10 10 10
Median 0.08 0.07 0.09 0.07
  Deviation Vs Control   -6.82 11.36 -9.09
BASOA Mean 0.01 k 0.01 0.01 0.01
[giga/L] S.d. 0.00 0.00 0.00 0.00
day 93 N 10 10 10 10
Median 0.01 0.01 0.01 0.01
  Deviation Vs Control   22.22 11.11 -22.22
LUCA Mean 0.01 k 0.01 0.01 0.01
[giga/L] S.d. 0.01 0.01 0.01 0.00
day 93 N 10 10 10 10
Median 0.01 0.01 0.01 0.01
  Deviation Vs Control   30.00 0.00 30.00
NEUT Mean 17.5 k 16.6 18.8 15.6
[%] S.d. 6.3 3.6 4.4 3.5
day 93 N 10 10 10 10
Median 15.9 15.6 18.2 16.1
  Deviation Vs Control   -5.5 7.0 -11.2
LYMPH Mean 76.7 k 78.3 75.5 79.9
[%] S.d. 8.5 4.1 5.4 4.0
day 93 N 10 10 10 10
Median 79.2 79.6 75.8 79.2
  Deviation Vs Control   2.2 -1.6 4.2
MONO Mean 2.0 k 2.0 2.3 2.0
[%] S.d. 0.8 0.5 0.3 0.3
day 93 N 10 10 10 10
Median 1.8 1.9 2.2 1.9
  Deviation Vs Control   3.5 13.6 1.0
EOS Mean 3.2 k 2.3 3.0 2.0
[%] S.d. 2.5 0.6 1.9 0.4
day 93 N 10 10 10 10
Median 2.5 2.1 2.3 1.9
  Deviation Vs Control   -28.5 -7.1 -39.3
BASO Mean 0.2 v 0.3 0.3 0.2
[%] S.d. 0.1 0.1 0.1 0.1
day 93 N 10 10 10 10
Median 0.2 0.3 0.3 0.2
  Deviation Vs Control   32.0 12.0 -24.0
LUC Mean 0.3 k 0.4 0.2 0.3
[%] S.d. 0.2 0.2 0.2 0.2
day 93 N 10 10 10 10
Median 0.3 0.4 0.2 0.3
  Deviation Vs Control   25.0 -25.0 9.4

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS; v=KRUSKAL-WALLIS-WILCOX

Enzymes

Males Dose group 0 1 2 3
ALT Mean 0.86 k 0.76 0.78 0.89
[µkat/L] S.d. 0.25 0.15 0.11 0.15
day 92 N 10 10 10 10
Median 0.79 0.81 0.79 0.87
  Deviation Vs Control   -11.68 -8.76 3.86
AST Mean 2.11 k 1.68 1.57 1.68
[µkat/L] S.d. 1.34 0.27 0.26 0.14
day 92 N 9 10 10 10
Median 1.68 1.68 1.50 1.69
  Deviation Vs Control   -20.42 -25.49 -20.66
ALP Mean 0.99 v 0.92 0.97 1.12*
[µkat/L] S.d. 0.13 0.14 0.22 0.14
day 92 N 10 10 10 10
Median 1.06 0.91 0.86 1.12
  Deviation Vs Control   -7.47 -2.53 12.73
GGT_C Mean 25 25 25 25
[nkat/L] S.d. 0 0 0 0
day 92 N 10 10 10 10
Median 25 25 25 25
  Deviation Vs Control   0 0 0

Statistic Profile = Wilcoxon test (one-sided+), Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from

statistics

k=KRUSKAL-WALLIS; v=KRUSKAL-WALLIS-WILCOX

Females Dose group 0 1 2 3
ALT Mean 0.60 v 0.59 0.78 ** 0.87 **
[µkat/L] S.d. 0.12 0.10 0.13 0.19
day 93 N 10 10 10 10
Median 0.57 0.59 0.77 0.82
  Deviation Vs Control   -1.84 30.27 46.15
AST Mean 1.54 k 1.49 1.49 1.42
[µkat/L] S.d. 0.13 0.27 0.25 0.23
day 93 N 10 10 10 10
Median 1.52 1.43 1.50 1.48
  Deviation Vs Control   -3.05 -3.31 -7.85
ALP Mean 0.52 k 0.45 0.55 0.52
[µkat/L] S.d. 0.13 0.07 0.13 0.10
day 93 N 10 10 10 10
Median 0.52 0.43 0.51 0.54
  Deviation Vs Control   -13.44 6.53 0.19
GGT_C Mean 25 x+ 25 25 27 *
[nkat/L] S.d. 0 0 0 3
day 93 N 10 10 10 10
Median 25 25 25 25
  Deviation Vs Control   0 0 8

Statistic Profile = Wilcoxon test (one-sided+), Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from

statistics

v=KRUSKAL-WALLIS-WILCOX; k=KRUSKAL-WALLIS; x=WILCOX

Substrates

Males Dose group 0 1 2 3
UREA Mean 5.63 k 5.28 5.41 5.60
[mmol/L] S.d. 0.74 0.59 0.45 0.77
day 92 N 10 10 10 10
Median 5.38 5.30 5.48 5.59
  Deviation Vs Control   -6.27 -3.96 -0.53
CREA Mean 25.9 k 25.8 26.7 25.3
[µmol/L] S.d. 2.6 2.4 2.0 2.5
day 92 N 10 10 10 10
Median 25.1 25.8 26.9 26.1
  Deviation Vs Control   -0.5 2.9 -2.4
GLUC Mean 6.29 k 6.39 6.07 5.72
[mmol/L] S.d. 0.87 0.49 1.14 0.87
day 92 N 10 10 10 10
Median 6.31 6.48 6.13 5.57
  Deviation Vs Control   1.65 -3.39 -8.94
TBIL_C Mean 1.37 v 1.43 1.20 * 0.97 **
[µmol/L] S.d. 0.19 0.22 0.13 0.17
day 92 N 10 10 10 10
Median 1.33 1.46 1.17 0.94
  Deviation Vs Control   4.43 -12.42 -29.12
TPROT Mean 62.01 k 63.02 63.38 63.89
[g/L] S.d. 2.22 2.37 1.77 1.11
day 92 N 10 10 10 10
Median 61.77 62.69 63.45 63.59
  Deviation Vs Control   1.62 2.20 3.03
ALB Mean 36.35 v 36.74 36.95 38.02 **
[g/L] S.d. 0.85 0.69 0.71 0.97
day 92 N 10 10 10 10
Median 36.53 36.88 36.92 38.13
  Deviation Vs Control   1.06 1.63 4.59
GLOB Mean 25.66 k 26.28 26.43 25.87
[g/L] S.d. 1.58 2.01 1.29 0.86
day 92 N 10 10 10 10
Median 25.66 26.14 26.47 26.10
  Deviation Vs Control   2.42 2.99 0.81
CHOL Mean 1.80 v 1.81 1.83 1.51 *
[mmol/L] S.d. 0.24 0.24 0.28 0.15
day 92 N 10 10 10 10
Median 1.79 1.71 1.82 1.50
  Deviation Vs Control   0.67 1.67 -16.16
TRIG Mean 1.36 k 1.34 1.29 0.86
[mmol/L] S.d. 0.55 0.58 0.48 0.30
day 92 N 10 10 10 10
Median 1.25 1.30 1.27 0.79
  Deviation Vs Control   -1.18 -5.01 -37.04

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS; v=KRUSKAL-WALLIS-WILCOX

Females Dose group 0 1 2 3
UREA Mean 6.60 k 7.03 7.51 7.19
[mmol/L] S.d. 1.01 1.16 0.91 1.15
day 93 N 10 10 10 10
Median 6.82 7.06 7.42 7.26
  Deviation Vs Control   6.51 13.72 8.89
CREA Mean 33.3 k 32.7 34.3 29.7
[µmol/L] S.d. 4.3 4.1 3.7 2.0
day 93 N 10 10 10 10
Median 31.5 32.0 34.7 30.7
  Deviation Vs Control   -1.7 3.1 -10.6
GLUC Mean 5.61 k 5.09 4.90 4.86
[mmol/L] S.d. 0.81 0.59 0.64 0.85
day 93 N 10 10 10 10
Median 5.45 5.06 5.18 4.92
  Deviation Vs Control   -9.32 -12.71 -13.32
TBIL_C Mean 1.90 v 1.80 1.55 1.07 **
[µmol/L] S.d. 0.47 0.33 0.25 0.16
day 93 N 10 10 10 10
Median 1.73 1.76 1.61 1.14
  Deviation Vs Control   -5.27 -18.31 -43.51
TPROT Mean 63.48 v 66.31 * 66.97 ** 68.31 **
[g/L] S.d. 2.10 3.47 2.31 2.17
day 93 N 10 10 10 10
Median 63.33 65.02 66.60 67.90
  Deviation Vs Control   4.46 5.49 7.60
ALB Mean 38.70 k 39.98 39.71 40.24
[g/L] S.d. 1.36 2.36 1.12 1.82
day 93 N 10 10 10 10
Median 38.09 39.25 39.55 39.98
  Deviation Vs Control   3.29 2.60 3.97
GLOB Mean 24.78 v 26.34 * 27.26 ** 28.07 **
[g/L] S.d. 0.98 1.39 1.27 1.27
day 93 N 10 10 10 10
Median 24.83 26.26 27.44 27.70
  Deviation Vs Control   6.29 10.00 13.27
CHOL Mean 1.47 k 1.49 1.53 1.57
[mmol/L] S.d. 0.31 0.25 0.31 0.20
day 93 N 10 10 10 10
Median 1.45 1.58 1.56 1.51
  Deviation Vs Control   1.50 4.36 6.95
TRIG Mean 0.96 k 0.78 0.79 1.15
[mmol/L] S.d. 0.48 0.23 0.30 0.51
day 93 N 10 10 10 10
Median 0.72 0.74 0.71 1.14
  Deviation Vs Control   -18.37 -17.54 20.46

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS; v=KRUSKAL-WALLIS-WILCOX

Electrolytes + minerals

Males Dose group 0 1 2 3
NA Mean 140.0 k  140.4 140.5 140.5
[mmol/L] S.d. 1.1 0.7 1.3 1.3
 day 92 N 10 10 10 10
Median 139.7 140.2 140.2 140.9
Deviation Vs Control 0.3 0.4 0.4
K Mean 4.95 k 4.83 4.80 4.75
[mmol/L] S.d. 0.23 0.28 0.20 0.25
day 92 N 9 10 10 10
Median 4.90 4.75 4.80 4.73
  Deviation Vs Control   -2.48 -3.03 -4.08
CL Mean 96.4 k 96.7 96.6 97.1
[mmol/L] S.d. 1.4 1.3 1.1 1.0
day 92 N 10 10 10 10
Median 96.2 96.5 96.7 97.4
  Deviation Vs Control   0.3 0.3 0.7
INP Mean 1.72 k 1.69 1.74 1.78
[mmol/L] S.d. 0.20 0.16 0.11 0.16
day 92 N 10 10 10 10
Median 1.75 1.68 1.72 1.83
  Deviation Vs Control   -1.63 0.81 3.60
CA Mean 2.56 k 2.58 2.61 2.59
[mmol/L] S.d. 0.06 0.07 0.05 0.05
day 92 N 10 10 10 10
Median 2.58 2.59 2.62 2.59
  Deviation Vs Control   0.58 1.91 1.09

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS

Females Dose group 0 1 2 3
NA Mean 138.1 k 137.8 137.9 137.3
[mmol/L]  S.d.  1.2 0.8 1.0 1.8
day 93 N 10 10 10 10
Median 137.9 137.4 138.0 137.8
  Deviation Vs Control   -0.2 -0.1 -0.6
K Mean 4.30 k 4.12 4.04 4.22
[mmol/L] S.d. 0.38 0.15 0.29 0.37
day 93 N 10 10 10 10
Median 4.32 4.10 3.96 4.18
  Deviation Vs Control   -4.25 -6.05 -1.98
CL Mean 96.4 v 95.6 95.7 94.5 **
[mmol/L] S.d. 1.2 1.4 1.4 1.6
day 93 N 10 10 10 10
Median 96.7 95.4 95.8 94.8
  Deviation Vs Control   -0.9 -0.8 -2.0
INP Mean 1.40 v 1.57 1.58 * 1.64 **
[mmol/L] S.d. 0.13 0.20 0.14 0.14
day 93 N 10 10 10 10
Median 1.46 1.56 1.60 1.62
  Deviation Vs Control   11.70 13.05 17.19
CA Mean 2.56 v 2.66 * 2.63 * 2.68 **
[mmol/L] S.d. 0.08 0.08 0.07 0.05
day 93 N 10 10 10 10
Median 2.54 2.64 2.65 2.68
  Deviation Vs Control   3.98 2.62 4.57

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS; v=KRUSKAL-WALLIS-WILCOX

Urinalysis

Males Dose group 0 1 2 3
PH_C Mean 6.4 k 6.5 6.3 6.5
[---] S.d. 0.5 0.3 0.5 0.2
day 89 N 10 10 10 10
  Median 6.5 6.5 6.5 6.5
PRO_C Mean 1 x+ 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 1 1 1 1
GLU_C Mean 0 x+ 0 0 0
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 0 0 0 0
KET_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 1 1 1 1
UBG_C Mean 1 x+ 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 1 1 1 1
BIL_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 1 1 1 1
BLOOD_C Mean 1 x+ 1 1 1
[---] S.d. 0 0 1 0
day 89 N 10 10 10 10
  Median 1 1 1 1
VOL Mean 4.5 k 4.4 4.5 4.9
[ml] S.d. 0.8 0.8 0.8 1.0
day 89 N 10 10 10 10
  Median 4.7 4.4 4.3 5.0
SP.GR._C Mean 1,073 k 1'072 1'085 1'083
[g/L] S.d. 16 14 9 11
day 89 N 10 10 10 10
  Median 1'074 1'074 1'083 1'083
CRYST_C Mean 2 x+ 2 2 3 *
[---] S.d. 1 1 1 0
day 89 N 10 10 10 10
  Median 2 2 2 3
RENAL EC_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 1 1 1 1
TRANS EC_C Mean 1 x+ 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 1 1 1 1
SQUAM EC_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 1 1 1 1
CASTS_C Mean 0 x+ 0 1 1 *
[---] S.d. 0 0 1 1
day 89 N 10 10 10 10
  Median 0 0 1 1
ERY_C Mean 1 x+ 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 1 1 1 1
LEUCO_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 10 10 10
  Median 1 1 1 1

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), Wilcoxon test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from

statistics

k=KRUSKAL-WALLIS; x=WILCOX

Females Dose group 0 1 2 3
PH_C Mean 5.8 k 5.4 5.7 6.1
[---] S.d. 0.7 0.5 0.7 0.6
day 89 N 10 8 10 10
  Median 6.0 5.0 5.5 6.5
PRO_C Mean 1 x+ 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1
GLU_C Mean 0 0 0 0
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 0 0 0 0
KET_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1
UBG_C Mean 1 x+ 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1
BIL_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1
BLOOD_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1
VOL Mean 3.3 k 2.9 3.0 3.5
[ml] S.d. 1.0 0.9 0.5 0.9
day 89 N 10 8 10 10
  Median 2.9 2.6 3.0 3.7
SP.GR._C Mean 1,087 k 1'093 1'085 1'084
[g/L] S.d. 18 19 9 16
day 89 N 10 8 10 10
  Median 1'087 1'096 1'087 1'082
CRYST_C Mean 2 x+ 2 2 2
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 2 2 2 2
RENAL EC_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1
TRANS EC_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1
SQUAM EC_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1
CASTS_C Mean 0 0 0 0
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 0 0 0 0
ERY_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1
LEUCO_C Mean 1 1 1 1
[---] S.d. 0 0 0 0
day 89 N 10 8 10 10
  Median 1 1 1 1

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), Wilcoxon test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from

statistics

k=KRUSKAL-WALLIS; x=WILCOX

Abbreviations used above:

HEMATOLOGY:

RBC = red blood cells (erythrocytes)

HGB = hemoglobin

HCT = hematocrit

MCV = mean corpuscular volume

MCH = mean corpuscular hemoglobin

MCHC = mean corpuscular hemoglobin concentration

RETA = reticulocytes (absolute)

PLT = platelets

HQT = prothrombin time (Hepato Quick's test)

PTT = activated partial thromboplastin time

QT = prothrombin time (Quick's test)

WBC = white blood cells (leukocytes)

NEUTA = polymorphonuclear neutrophils (absolute)

LYMPHA = lymphocytes (absolute)

MONOA = monocytes (absolute)

EOSA = eosinophils (absolute)

BASOA = basophils (absolute)

LUCA = large unstained cells (absolute)

NEUT = polymorphonuclear neutrophils

LYMPH = lymphocytes

MONO = monocytes

EOS = eosinophils

BASO = basophils

LUC = large unstained cells

CLINICAL CHEMISTRY:

ALT = alanine aminotransferase

AST = aspartate aminotransferase

ALP = alkaline phosphatase

GGT_C = serum-γ-glutamyltransferase

UREA = urea

CREA = creatinine

GLUC = glucose

TBIL = total bilirubin

TBA = bile acids

TPROT = total protein

ALB = albumin

GLOB = globulins

TRIG = triglycerides

CHOL = cholesterol

NA = sodium

K = potassium

CL = chloride

INP = inorganic phosphate

CA = calcium

URINALYSIS:

pH/pH_C = pH value

PRO = protein

0 = negative 0.25 g/L

1 = 0.75 g/L

2 = 1.5 g/L

3 = 5.00 g/L

PRO_C = for statistical calculation grade 0 + 1 is set grade 1

GLU = glucose

0 = normal

1 = 3 mmol/L

2 = 6 mmol/L

3 = 17 mmol/L, 56 mmol/L

GLU_C = glucose

KET = ketones

0 = negative

1 = 0.5 mmol/L, 1.5 mmol/L

2 = 5 mmol/L

3 = 15 mmol/L

KET_C = for statistical calculation grade 0 + 1 is set grade 1

UBG = urobilinogen

0 = normal

1 = 17 μmol/L

2 = 68 μmol/L

3 = 135 μmol/L, 203 μmol/L

UBG_C = for statistical calculation grade 0 + 1 is set grade 1

BIL = bilirubin

0 = negative

1 = 17 μmol/L

2 = 50 μmol/L

3 = 100 μmol/L

BIL_C = for statistical calculation grade 0 + 1 is set grade 1

BLOOD = blood (erythrocytes)

0 = negative

1 = 10 ery/μL

2 = 25 ery/ μL, 50 ery/μL

3 = 150 ery/μL, 250 ery/μL

BlOOD_C = for statistical calculation grade 0 + 1 is set grade 1

VOL = volume

SP.GR._C = specific gravity calculated with dilution factor

TURB = turbidity

c = clear

cl = cloudy

COL = color

y = yellow

dyl = dark yellow

ly = light yellow

SEDIMENT:

CRYST = crystals

RENAL EC = renal epithelial cells

TRANS EC = transitional epithelial cells

SQUAM EC = squamous epithelial cells

CASTS = casts

ERY = erythocytes

LEUCO = lymphocytes

0 = none

1 = few

2 = many

3 = masses

CRYST_C = for statistical calculation grade 0 - 2 is set grade 2

RENAL

EC_C

= for statistical calculation grade 0 + 1 is set grade 1

TRANS

EC_C

= for statistical calculation grade 0 + 1 is set grade 1

SQUAM

EC_C

= for statistical calculation grade 0 + 1 is set grade 1

CASTS_C = casts

ERY_C = for statistical calculation grade 0 + 1 is set grade 1

LEUCO_C = for statistical calculation grade 0 + 1 is set grade 1

P = triple phosphates

O = calcium oxalates

C = crystals of unknown origin

T = tyrosine-like crystals

GCE = granulated casts/epithelial casts

CE = epithelial casts

V = clusters

a = macrohematuria

b = slight macrohematuria

c = epithelial scaling

d = atypical epithelial cells

e = degenerated epithelial cells

f = squamous epithelial cells with some nuclei

g = transitional epithelial cells with some nuclei

h = caudate epithelial cells with some nuclei

HISTORICAL CONTROL DATA OF CLINICAL PATHOLOGY TESTING

MALE
Parameter: MONO MONO ALP ALB
Unit: % GIGA/L MYKAT/L G/L
N (wo. outliers) 57 56 57 57
Mean 2.1 0.11 1.17 36.66
25. Percentile (Q25) 1.8 0.10 1.10 34.90
75. Percentile (Q75) 2.2 0.12 1.24 38.43
Q25-1.5x(Q75-Q25) 1.2 0.07 0.89 29.61
Q75+1.5x(Q75-Q25) 2.8 0.15 1.45 43.73
Minimum (wo. outliers) 1.6 0.08 0.97 33.28
Maximum (wo.outliers) 2.8 0.15 1.35 40.67

*Outliers: < Q25-1.5x(Q75-Q25) or > Q75+1.5x(Q75-Q25)

FEMALE
Parameter: CA TPROT GLOB
Unit: MMOL/L G/L G/L
N (wo. outliers) 55 58 58
Mean 2.58 66.31 26.18
25. Percentile (Q25) 2.54 65.27 24.44
75. Percentile (Q75) 2.60 67.38 28.01
Q25-1.5x(Q75-Q25) 2.44 62.09 19.09
Q75+1.5x(Q75-Q25) 2.71 70.55 33.35
Minimum (wo. outliers) 2.50 63.21 21.63
Maximum (wo.outliers) 2.69 70.49 32.75

*Outliers: < Q25-1.5x(Q75-Q25) or > Q75+1.5x(Q75-Q25)

PATHOLOGY

Relevant mean percent liver weight differences from control

Sex:

Males Females

Dose in mg/kg bw/d:

10 50 250 10 50 250

Liver weight

           

Mean Absolute:

105 113* 127** 97 113* 149**

Mean Relative:

103 114** 134** 101 111* 152**

* = p ≤ 0.05, ** = p ≤ 0.01. Test item-related changes are presented in bold.

Mean liver weights were dose-relatedly increased in males and females of the 50 and 250 mg/kg bw/d treated group. For both sexes the increase in weight was minimal at 50 mg/kg b/d, while at 250 mg/kg bw/d it was considered slight in males and moderate in females. In the livers of animals treated with 250 mg/kg different microscopic changes were recognized: centrilobular hepatocellular hypertrophy, intracytoplasmic pigment accumulation and (peri)vasculitis. Centrilobular hepatocellular hypertrophy was observed among females in the group treated daily with 50 mg/kg bw/d and in males and females treated with 250 mg/kg. Hepatocellular hypertrophy is believed to represent the morphologic hallmark of enzyme induction. This change resulted in increased mean liver weights in males and females of the mentioned groups. It is considered an adaptive non-adverse response, if not seriously present and if not accompanied by increases in liver enzymes of a factor two or higher (Hall et al., 2012). However, in case of the mean liver weight increase in females of the 250 mg/kg bw/d treated group, that exceeded 40% it is regarded an adverse change. The less severe changes in males and females of the other groups are considered a non-adverse adaptive response.

A second test item-related finding related to the hepatocytes of animals treated with 250 mg/kg consisted of the appearance of a fine brown intracellular pigment within centrilobular located hypertrophied hepatocytes. Females were more affected compared to males. Using different staining methods to characterize the pigment, it was proven to consist of lipofuscin mainly (the pigment was positive for Schmorl and PAS, while the Hall’s and Perl’s staining methods were negative). Possibly both changes are related, since this change could point to the formation of reactive metabolite(s) that on its turn could react with cellular structures such as membranes (lipid peroxidation) resulting in the formation of lipofuscin containing pigment. The dark brown discoloration of livers from animals treated with 250 mg/kg bw/d could not completely be explained by the presence of lipofuscin, since, especially in males the number of dark brown discolored livers exceeded the number of livers with (visible) intracytoplasmic pigment accumulation. Further, the amount of pigment was not excessive. Because of the underlying mechanism, the formation of lipofuscin is believed to represent an adverse finding.

(Peri)vasculitis related to the branches of the hepatic artery was observed at a relative high incidence (30%) in males of the 250 mg/kg bw/d treated group. The vascular change had similar characteristics in all animals with degeneration and fibrinoid necrosis of the vessel wall and a perivascular inflammatory infiltrate (macrophages mainly) and fibroblast reaction. The observed percentage of 30% is comparable to the highest incidence of (peri)vasculitis observed among control male rats: historical control data related to this strain of rats includes incidences of (peri)vasculitis in the liver that varies between 0% and 30% in male control rats, while the lesion was not recorded in control female rats. Because the lesion was also found in a single male and a single female treated with 50 mg/kg bw/d, and a precursor lesion was observed in a single female treated with 250 mg/kg bw/d, a relationship to the treatment with the test item cannot be ruled out. Possibly, the test item aggravated this spontaneous occurring lesion. In a single female of the group treated with 50 mg/kg bw/d an area of slight hepatocellular necrosis was found to be closely related to focal vasculitis.

Relevant mean percent adrenal gland weight differences from control

Sex Males Females
Dose in mg/kg bw/d: 10 50 250 10 50 250
Adrenal weight
Mean Absolute: 105 102 108 99 112 124**
Mean Relative: 102 103 113 103 109 126**

* = p ≤ 0.05, ** = p ≤ 0.01.

The weight changes noted for the adrenal gland in females of the 250 mg/kg bw/d treated group were not supported by obvious histopathologic changes. Most probably the increase adrenal gland weight can be explained by a diffuse increased cortical cell size that could not be recognized at light microscopic level. Since there were no relevant changes noted in the adrenal glands, the weight changes are considered non-adverse and of no significant toxicologic relevance.

Relevant mean percent ovary weight differences from control

Sex Males Females
Dose in mg/kg bw/d: 10 50 250 10 50 250
Ovarian weight
Mean Absolute: 89 105 118*
Mean Relative: 92 103 120*

* = p ≤ 0.05, ** = p ≤ 0.01.

A slight, statistically significant increase of the mean absolute and relative ovarian weight was present in females treated with 250 mg/kg bw/d test item. Microscopically, 6 out of 10 females of this group exhibited minimal interstitial cell vacuolation. This interstitial cell vacuolation was at least partial responsible for the increase in ovarian weight. In theory, vacuolation of interstitial cells may be associated with alterations of steroid synthesis leading to lipid accumulation within the cells. This change however is of questionable adversity, since, based on the ovarian morphology, it does not seem to have had influence on the ovarian function and the cyclicity seems not to be disturbed in high-dose females.

Relevant mean percent thyroid weight differences from control

Sex Males Females
Dose in mg/kg bw/d: 10 50 250 10 50 250
Thyroid weight
Mean Absolute: 99 102 117 85* 99 108
Mean Relative: 96 102 122* 88 97 110

* = p ≤ 0.05, ** = p ≤ 0.01.

A minimal increase in mean thyroid gland weight was observed in male animals of the 250 mg/kg bw/d treated group, only. This weight change was not supported by convincing histopathologic changes (only one male showed minimal follicular hypertrophy worth mentioning). The only finding that could point to a treatment-related effect is the presence of a slight higher incidence of colloid changes (inspissated colloid) in several high-dose males. Nevertheless, both the changes in thyroid gland weight and histology are considered of uncertain test-item relationship.

HISTOPATHOLOGY INCIDENCE TABLE: TREATMENT-RELATED FINDINGS

Dose Group 0 1 2 3
Sex M F M F M F M F
No of animals 10 10 10 10 10 10 10 10
LIVER 10 10 10 10 10 10 10 10
Centril.Hypertrophy  - - - - - 7 10 10
Grade 1 - - - - - 5 2 -
Grade 2 - - - - - 2 8 1
Grade 3 - - - - - - - 9
Pigment - - - - - - 4 8
Grade 1 - - - - - - 4 2
Grade 2 - - - - - - - 6
Degen./Necr. Vascular - - - - 1 1 3 1
Grade 1 - - - - - - 1 -
Grade 2 - - - - - 1 1 -
Grade 3 - - - - 1 - 1 -
(Peri)vasculitis - - - - 1 1 3 -
Grade 1 - - - - - - 2 -
Grade 2 - - - - 1 - 1 -
Grade 3 - - - - - 1 - -
Necrosis - - - - 1 - - -
Grade 2 - - - - 1 - - -
OVARIES - 10 - - - - - 10
Incr.vacuol.Int.cell - - - - - - - 6
Grade 1 - - - - - - - 6

HISTOPATHOLOGY INCIDENCE TABLE: ALL FINDINGS OF RELEVANT ORGANS

Dose Group 0 1 2 3
Sex M F M F M F M F
No. Of animals 10 10 10 10 10 10 10 10
LIVER 10 10 10 10 10 10 10 10
N.A.D. 6 6 9 9 7 3
Infiltr. Inflamm 4 4 1 1 3 3 1
Grade 1: 4 3 1 1 3 3 1
Grade 2: 1
Centril.Hypertrophy: 7 10 10
Grade 1: 5 2
Grade 2: 2 8 1
Grade 3: 9
Single Cell Necrosis: 1
Grade 1: 1
Pigment: 4 8
Grade 1: 4 2
Grade 2: 6
Aggr.pigment.macroph: 1
Grade 1: 1
Degen/Necr. vascular: 1 1 3 1
Grade 1: 1 1
Grade 2: 1 1
Grade 3: 1 1
(Peri)vasculitis: 1 1 3
Grade 1: 2
Grade 2: 1 1
Grade 3: 1
Necrosis: 1
Grade 2: 1
THYROID GLAND 10 10 10 10
N.A.D. : 7 9 3 1
Colloid alteration: 1 3 1
Grade 1: 1 3 1
C-Cell Hyperpl/Focal: 1
Grade 1: 1
Hypertr. follic.cell: 1
Grade 1: 1
Ectopic thymus tiss.: 2 1
Ultimobranchial Cyst: 1 1 5 1
ADRENAL GLANDS 10 10 10 10
N.A.D. : 9 10 9 10
Vacuol. cortic. cell: 1
Grade 2: 1
Mineralization: 1
Grade 1: 1
Incr.vacuol.z.glom. : 1
Grade 2: 1
OVARIES 10 10 10 10
N.A.D. : 10 10 10 4
Incr.vacuol.Int.cell: 6
Grade 1: 6
KIDNEYS 10 10 10 1 10 10 10
N.A.D. : 1 2 5
Nephroblastematosis: 1
Grade 1: 1
Cyst(s): 1 1 3
Hyaline dropl. CAB+: 9 8 10 10
Grade 1: 8 4 6 3
Grade 2: 1 4 4 6
Grade 3: 1
Basophilia tubule: 3 3 5 4 5
Grade 1: 3 1 5 4 3
Grade 2: 2 2
Fibrosis: 1 1
Grade 1: 1 1
Artery:medial hypert: 1
Grade 2: 1
Infiltr. inflamm.: 1 4
Grade 1: 1 3
Grade 2: 1
Infarct: 1
Grade 2: 1
Dilation pelvis     1
 Grade 2: 1
Dilation tubule     1 2 1 2
Grade 1: 1 2 1 2
Mineralization      4 2 3
Grade 1: 3 2 1
Grade 2: 1 2

Findings in the kidney:

Although there is a broad variation in the severity of hyaline droplet accumulation in the kidney of male rats in general, for the slight increase in severity observed in male rats of the 250 mg/kg bw/d treated group compared to the control males in this study a relationship to treatment cannot be excluded. The hyaline droplets were proven to consist of alpha2U-globulin mainly.

Based on the pathology, the No-Observed-Adverse-Effect-Level (NOAEL) is 50 mg/kg.

HISTORICAL CONTROL DATA (PERI)VASCULITIS IN THE LIVER

MALES Animal Number (Peri)Vasculitis in the liver
Number (%)
Sum: 120 5  
Mean:     4.2
Number of Studies: 12    
Maximum:     30
Minimum:     0
MALES Animal Number (Peri)Vasculitis in the liver
Number (%)
Sum: 120 0  
Mean:     0.0
Number of Studies: 12    
Maximum:     0
Minimum:     0

In the historical control data, the incidence of (Peri)Vasculitis in males varied between 0% and 30%.

In the historical control data, the incidence of (Peri)Vasculitis in females was 0%.

Applicant's summary and conclusion

Conclusions:
The oral administration of the test item by gavage to male and female Wistar rats for 3 months caused adverse signs of toxicity in male and female animals at a dose level of 250 mg/kg bw/d taking clinical pathology and pathology findings into account. Therefore, the no observed adverse effect level (NOAEL) was set to 50 mg/kg bw/d for male and female Wistar rats.
Executive summary:

The test article was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 10 (test group 1), 50 (test group 2) and 250 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months. Polyethylene glycol (PEG 400) served as vehicle. In addition to the required examinations, special attention was given to the reproductive organs of male and female animals. Food consumption and body weight were determined weekly. Water consumption was determined once a week from study day 33 onwards. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. For at least 3 weeks an estrous cycle determination was performed. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinico-chemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations. Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals for sperm examinations.

During clinical examinations, the following observations were made in high dose animals: prolonged prothrombin time in males, decreased cholesterol levels in males, increased γ-glutamyl transferase (GGT) activities in females and increased inorganic phosphate levels in females. During pathology increased absolute (males +27%, females +49%) and relative (males +34%, females +52%) liver weights were recorded. Dark brown discoloration of livers in 7/10 males and 8/10 females were reported. In addition, minimal to moderate centrilobular hepatocellular hypertrophy of the livers in all male and female animals and minimal to slight pigment accumulation within hepatocytes in male (4/10) and female (8/10) animals were observed. No treatment-related, adverse effects were observed for animals of the mid and low dose groups. In conclusion, the oral administration of the test item by gavage to male and female Wistar rats for 3 months caused adverse signs of toxicity in male and female animals at a dose level of 250 mg/kg bw/d taking clinical pathology and pathology findings into account. Therefore, the no observed adverse effect level (NOAEL) was set to 50 mg/kg bw/d for male and female Wistar rats.