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OECD 422

Daily oral administration of the test substance to Wistar rats at dose levels of 111, 333, and 1000 mg/kg body weight over a period of 44 to 57 days resulted at the highest dose level in a reduction of the body weight gain and an increased water and food consumption in male animals. The water consumption was increased in females of the high dose group. Test item related changes in organ weight were found in the liver, the kidney and thymus of male animals. Furthermore, in male rats the histopathological examination indicated a nephrotoxic effect in all dose groups. At the highest dose level, the test item also showed a potential effect on the metabolism of the liver as well as an influence on uterus weight of female rats.

The NOAEL is 333 mg/kg bw for females. In males based on the effects on the kidney a LOAEL of 111 mg/kg bw was derived. As these effects are considered of less relevance for humans, the NOAEL used in risk assessment is set at 333 mg/kg bw.

OECD 408

Male and female rats (10/sex/dose) were treated with the substance at 0, 100, 300 and 1000 mg/kg bw by gavage for 90 days according to OECD 408. No treatment findings on mortality, food intake and haematology were noted. At 1000 mg/kg bw liver and kidney weights were increased in males and females. In animals at 1000 mg/kg bw a time-related increased incidence of piloerection, decreased motility and laying in a rigid position was seen lasting up to 1.5 hours after dosing from day 28 onwards.

In females at 1000 mg/kg bw body weight (gain) was within control ranges , but slight effects on biochemistry (total protein (globulin), ALP, bile acids and cholesterol) were observed. Liver and kidney weights were increased.

Macroscopic and histopathological evaluation of these females showed enlarged and pale kidneys with hyaline casts. The lungs showed aspired blood and focal lipidosis. The NOAEL for females is therefore set at 300 mg/kg bw.

Males at 1000 mg/kg bw had a decreased body weight (gain). In addition these males showed decreased blood alkeline phosphatase levels and increased urinary ketone bodies and protein. At 300 and 1000 mg/kg kidney and liver weights increased in a dose dependent way. This was not accompanied by macroscopic and histological findings in the liver, but kidneys were found to be enlarged/pale containing hyaline droplets, hyaline casts, tubular basophilia and tubular necrosis indicative of chronic progressive nephrosis. Similar effects in the kidney were observed in all males at 100 and 300 mg/kg bw. These effects were confirmed to be related to α2-microglobulin (immunohistochemistry) and are therefore not relevant to humans. Effects in the lungs were similar to those found in females. Based on these findings no NOAEL could be set for males.

The NOAEL for females will be used as starting point for risk assessment.

Based on the information above and the absence of effects in a study according to OECD 414 at 1000 mg/kg bw (highest dose tested), the NOAEL that will be used as starting point for the risk assessment is set at 300 mg/kg bw. It is expected that this value is sufficiently protective for potential effects on fertility.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 November 2015 to 15 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain:Rattus norvegicus
- Source: MediTox s.r.o., The Czech Republic
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 7-9 weeks (6-7 weeks at delivery)
- Weight at study initiation: males 206-268 g; females 174-214 g
- Fasting period before study: none
- Housing: Macrolon Tecniplast cages (2-3 animals of the same sex/cage)
- Diet: “Myši chov” (Sehnoutek a synové v.o.s., Czech Republic) ad libitum
- Water : ad libitum
- Acclimation period: 8-11 days

DETAILS OF FOOD AND WATER QUALITY: available at the lab

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): not indicated (ventilated room)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: weekly in corn oil using magnetic stirring

VEHICLE corn oil
- Justification for use and choice of vehicle (if other than water): similar to reproduction/developmental toxicity screening test for Paramenthan (OECD 422)”, study report I 16-001, vivo Science GmbH, Germany.
- Concentration in vehicle: 0, 20, 60 and 200 mg/L
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: MKBV2080V
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Three samples from were taken from top middle and bottom of the dosing formulation in week 1 and week 13 of the study (December 09, 2015, and February 29, 2016). Samples were transported to Toxi-Coop ZRT and analysed on the day after arrival (December 16-17, 2015 and March 03-04, 2016).
During transport there was a deviation from the preferred storage temperature, but in view of the outcome of the analyses the stability of the substance has not been affected.

Samples were diluted with n-hexane in duplicate except for the control. A volume of 0.5 mL of the internal standard solution was added before the samples were filled up to 10 mL final volume.

GC/FID
Column: Rtx-502.2, 60 m x 0.25 mm x 1.4 µm, No: 1023994
Temperature program: 125°C to 150°C at 2°C/min
2 min hold
150°C to 180°C at 20°C/min
2 min hold
Injector: 200°C
Detector: FID, 200°C
Carrier gas: nitrogen, 2 mL/min, constant flow
Split ratio: 20
Injection volume: 0.5 µL
Retention times: para-Menthane 13.5-13.6 and 14.4-14.5 min
1-Octanol: 16.9-17.0 min
Evaluation: Sum of the areas of para-Menthane peaks is calculated and divided by 1-octanol peak area. This ratio is used for the computations.

Linearity over 0.1 – 3 mg/mL (r2 = 0.999 and 1.000) based on 6 calibration standard solutions
Recovery in corn oil: 96% at 1 mg/L; 95% at 500 mg/L
Stability: 14 days
LOQ: 0.1 mg/L
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle controls
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
measured 93-95% of nominal
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
measyred 94-96% of nominal
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
measured 100-101% of nominal
No. of animals per sex per dose:
10 males + 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of the reproduction/developmental toxicity screening test for Paramenthan (OECD 422)”, study report I 16-001, vivo Science GmbH, Germany.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly in standard arena: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: at start and weekly thereafter

FOOD CONSUMPTION: Yes
- Food consumption: weekly calculated as g food/kg body weight/day:

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and in week 13
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-treatment and week 13
- Anaesthetic used for blood collection: Yes slight ether anesthesia
- Animals fasted: Yes 12-18 hours before sampling
- How many animals: all
- Parameters checked: Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Leukocyte count (WBC), Platelet count (PLT), Nucleated RBC, Differential leukocyte count, Activated partial thromboplastin time (APTT), Prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre-treatment and week 13
- Anaesthetic used for blood collection: Yes slight ether anesthesia
- Animals fasted: Yes 12-18 hours before sampling
- How many animals: all
- Parameters checked: Alanine aminotransferase, Albumin, Albumin/Globulin ratio, Alkaline phosphatase, Aspartate aminotransferase, Bile Acids, Bilirubin total, Calcium, Chloride, Cholesterol, Creatinine, Gamma-Glutamyl Transpeptidase, Glucose, Globulin, Lactate Dehydrogenase, Phosphorus, Potassium, Sodium, Total protein, Triglycerides, Urea

URINALYSIS: Yes
- Time schedule for collection of urine: pre-treatment and in week 13
- Metabolism cages used for collection of urine: Yes animals kept in metabolism cages for an interval of two hours after previous oral administration of water in volume of 2 mL/100 g of b.w.
- How many animals: all
- Animals fasted: No
- Parameters checked: Volume, Appearance, color,Specific gravity, pH, Protein, Bilirubin, Urobilinogen, Blood, Ketones, Nitrite, Leucocytes, Glucose, Turbidity

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table) on all animals

ORGAN WEIGHTS: : Yes (see table) of all animals

HISTOPATHOLOGY: Yes (see table) of all animals in the control and high dose group + kidneys and lungs of the intermediate groups, organs preserved in 4 % neutral buffered formaldehyde. The eyes, optic nerves, testes and epididymides were fixed in Davidson's fluid.
Other examinations:
Renal Immunohistochemistry
in males only: the rationale for these investigations was to demonstrate that the hyaline droplets observed in a previous study consist of α2µ globulin. Hyaline droplets are sex specific and are observed in male rats only.
Wax embedded samples of the right kidney of all males were taken and transported to AnaPath (Switzerland). Samples underwent immunohistochemistry with Rat α2µ-Globulin Antibody (Monoclonal Mouse IgG1 Clone #129736, R&D Systems). The image analysis was performed by the CELL Analysis System. Pictures of kidney slides were taken by an Olympus UC30 camera at a magnification of x20. The positive area on the total area were measured in mm2 and calculated as % of the total area. The relative values of positive structures (α2µ-globulin) were used for descriptive statistics (mean, standard deviation, minimum, maximum).
Statistics:
The analyzed data were tested for normality (Kolmogorov–Smirnov test) and homogeneity of variance (Bartlett’s test or F test).
The data for males and females were separately analyzed using ANOVA followed by Dunnett’s Multiple Comparison Test.
In addition if necessary Kruskal–Wallis test (which compares medians instead of means) followed by Dunn’s Multiple Comparison were used.
Absolute and relative organ weight values for test article dosed groups were compared to those of control using the Student’s t Test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the acclimation period, no clinical signs were recorded in any of the animals in the study.
No clinical signs of toxicity were observed during the treatment period in the animals from the control, low-dose (D1), and the middle-dose (D2) groups.
All males in the group D3, except one male (M36), and four D3 females (F72, F75, F76, F79) showed repeated occurrence of piloerection, mark decrease of motility, these rats lied in rigid position. These symptoms appeared always 10 minutes after the administration and disappeared after 1-1.5 hours. These clinical findings started from day 28 of the Test Item administration and continued to the end of administration period.

The detailed clinical examination performed weekly did not reveal any another clinical symptoms of toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean and individual body weight values of males and females in all groups increased during the administration period.
Statistically significant decrease in the mean body weight by 10-12 % was found in males from the high dose group (D3) from week 9 to the end of administration period compared to the controls. However, insignificant dose depended decrease of the body weight was observed in males from the D2 and D1 dose group.
No statistically significant difference of the body weight was recorded in females in all groups during the administration period.
Detailed information about the body weight developments are given in the table below or can be found in the attached tables in pdf format.
The described decrease of the body weight in males was caused by the Test Item administered.

Body weight - mean, median and SD values
Tab.: 5
Males
Group Day At delivery -1 1 2 3 4 5 6 7 8 9 10 11 12 13
C
ANOVA p 0,999 0,132 0,659 0,959 0,930 0,283 0,289 0,223 0,099 0,056 0,037 0,037 0,019 0,012 0,006
K-W test p 0,996 0,209 0,769 0,983 0,929 0,159 0,217 0,169 0,118 0,063 0,033 0,033 0,025 0,021 0,010
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 188,8 236,2 296,2 337,5 372,8 400,8 430,2 451,6 470,4 487,9 508,5 521,8 537,0 550,6 558,9
Median 190,0 235,0 299,0 340,5 378,0 410,0 439,0 461,0 477,5 490,5 516,5 535,0 544,5 557,5 572,0
SD 10,9 12,8 15,3 19,9 18,6 23,2 24,0 27,5 27,8 31,7 31,6 33,3 33,9 34,2 34,9
D1
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 188,0 244,8 297,4 338,4 369,5 392,4 417,6 442,2 460,0 476,2 492,2 502,5 517,2 529,8 535,7
Median 190,0 246,0 300,0 341,0 373,0 395,0 421,0 445,0 459,5 475,0 487,0 499,5 514,5 525,0 533,0
SD 11,8 14,8 18,7 24,9 29,8 30,9 32,5 36,2 38,6 39,0 42,9 43,7 45,1 45,7 46,2
D2
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 188,8 245,6 295,8 335,6 368,9 382,6 412,0 431,0 446,0 461,7 475,8 485,8 499,4 508,1 514,7
Median 188,0 247,0 295,0 334,0 367,5 389,0 414,0 432,0 444,5 465,0 478,0 488,5 505,5 515,5 525,5
SD 12,4 18,2 16,9 20,7 23,9 22,1 28,0 30,8 34,4 37,4 36,5 39,6 41,2 39,1 41,4
D3 *** *** *** *** ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P < 0.05 P < 0.01 P < 0.01 P < 0.01
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.01
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 188,6 231,6 288,6 324,1 357,6 379,5 405,6 423,0 433,4 444,7 460,1 469,7 478,8 489,9 489,7
Median 189,0 232,0 296,0 329,0 359,0 382,0 406,0 424,0 434,5 443,0 455,0 467,5 478,0 489,5 489,5
SD 11,5 14,5 17,7 27,2 28,3 29,5 30,7 33,2 34,4 33,6 36,8 42,1 40,9 43,2 45,1
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)
# no variability in one of the columns

Tab.: 6
Females
Group Day At delivery -1 1 2 3 4 5 6 7 8 9 10 11 12 13
C
ANOVA p 1,000 0,820 0,197 0,621 0,970 0,992 0,955 0,864 0,524 0,773 0,753 0,815 0,634 0,933 0,921
K-W test p 1,000 0,867 0,210 0,760 0,981 0,979 0,993 0,905 0,619 0,955 0,966 0,905 0,844 0,975 0,970
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 159,6 194,0 215,6 237,9 255,4 266,4 273,9 285,4 289,1 295,3 301,0 307,7 313,7 316,1 323,4
Median 158,0 195,0 215,0 239,0 261,0 267,5 276,5 288,5 288,5 297,5 299,0 310,5 313,5 316,0 325,0
SD 7,2 10,5 10,9 16,2 19,5 18,7 17,0 22,0 22,8 20,5 20,8 27,4 28,4 24,2 27,7
D1
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 159,6 198,2 228,0 240,4 256,0 268,0 274,9 280,5 285,4 290,3 296,6 301,6 304,6 315,5 321,3
Median 158,0 201,0 234,0 244,0 263,0 275,0 282,0 287,5 292,0 298,5 307,0 306,5 313,0 317,0 327,5
SD 6,8 12,6 13,9 14,8 18,0 18,5 19,9 17,4 17,1 19,0 21,0 18,7 19,6 14,5 17,4
D2
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 159,6 196,6 224,8 244,0 257,2 268,2 277,1 285,6 294,7 296,6 304,0 308,8 314,0 319,2 326,3
Median 159,0 197,0 220,0 241,0 253,0 268,0 279,0 281,5 295,0 298,0 303,5 302,5 313,0 319,5 326,5
SD 7,0 7,9 13,9 9,9 11,3 10,3 11,9 11,0 11,9 9,4 13,1 14,5 12,5 12,1 14,0
D3
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 159,6 196,6 224,8 244,0 257,2 268,2 277,1 285,6 294,7 296,6 304,0 308,8 314,0 319,2 326,3
Median 159,0 197,0 220,0 241,0 253,0 268,0 279,0 281,5 295,0 298,0 303,5 302,5 313,0 319,5 326,5
SD 7,0 7,9 13,9 9,9 11,3 10,3 11,9 11,0 11,9 9,4 13,1 14,5 12,5 12,1 14,0
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)
# no variability in one of the columns

Individual data are attached as pdf file.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Several statistically significant differences of the mean food consumption were found in all treated males and females during administration period compared to control animals. There was no correlation between the food consumption and decrease of body weight in males of all dose groups.
No effect of the Test Item administration on food consumption was recorded.

Food consumption - mean, median and SD values
Tab.: 5
Males
Group Day -1 1 2 3 4 5 6 7 8 9 10 11 12 13
C + + + +
ANOVA p P<0.0001 0,024 0,837 0,471 0,002 P<0.0001 P<0.0001 0,022 0,018 0,081 0,287 0,313 P<0.0001 0,237
K-W test p 0,000 0,013 0,846 0,314 0,013 P<0.0001 P<0.0001 0,052 0,109 0,038 0,353 0,204 0,000 0,710
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 182,4 193,1 199,6 198,5 211,7 224,2 187,1 172,0 190,0 184,5 190,3 198,1 175,7 140,2
Median 179,5 191,0 198,0 198,5 215,0 222,0 187,5 172,5 190,5 183,5 188,5 198,5 177,0 144,0
SD 8,6 6,9 17,3 8,2 6,3 4,3 2,7 11,3 7,7 9,3 6,2 2,4 5,9 10,9
D1 *** *** ** ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P < 0.01 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 191,8 186,1 199,0 196,4 203,1 202,0 202,9 178,7 190,0 195,0 196,3 196,6 194,7 145,7
Median 194,0 184,5 199,0 199,0 201,5 202,0 202,5 177,5 189,0 192,0 192,5 194,0 195,5 145,0
SD 8,8 8,0 6,7 9,0 5,8 6,7 3,9 5,3 3,7 9,5 11,3 7,8 5,1 4,1
D2 * *** *** *** * ***
Dunnet's MC test P < 0.05 P < 0.01 P > 0.05 P > 0.05 P < 0.01 P < 0.01 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05
Dunn's MC test P > 0.05 P < 0.01 P > 0.05 P > 0.05 P < 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 195,7 180,9 194,6 193,3 197,5 197,2 185,0 176,2 181,4 190,5 186,6 201,6 191,4 145,6
Median 202,0 178,5 194,0 190,0 198,0 197,5 184,5 176,5 183,5 189,0 184,5 201,0 193,0 145,0
SD 17,7 9,7 12,3 6,8 9,2 1,7 4,2 13,1 9,2 9,6 13,7 5,2 5,7 5,9
D3 * *** *** ***
Dunnet's MC test P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 169,5 186,5 195,4 197,4 208,6 206,1 183,6 188,3 191,0 196,3 196,1 203,4 189,7 148,5
Median 166,0 185,0 196,0 199,0 202,0 202,0 183,0 181,0 192,0 187,0 188,0 195,0 189,0 145,0
SD 8,5 8,7 20,0 6,2 9,8 10,4 5,1 14,2 7,4 14,1 18,1 14,9 10,7 12,5
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)
# no variability in one of the columns


Tab.: 6
Females
Group Day -1 1 2 3 4 5 6 7 8 9 10 11 12 13
C + + + + + +
ANOVA p 0,698 0,380 P<0.0001 0,129 P<0.0001 0,034 P<0.0001 P<0.0001 P<0.0001 P<0.0001 P<0.0001 0,002 P<0.0001 0,003
K-W test p 0,779 0,439 P<0.0001 0,304 P<0.0001 0,104 0,000 P<0.0001 P<0.0001 0,000 P<0.0001 0,004 0,000 0,003
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 147,2 148,1 153,2 150,7 165,2 139,3 144,9 137,4 157,5 148,3 156,1 152,0 127,5 135,4
Median 147,0 148,5 152,5 151,5 165,0 140,0 142,5 137,0 157,5 147,5 154,0 153,0 128,0 123,5
SD 2,5 6,4 2,7 8,0 5,0 4,9 5,3 5,4 4,1 5,5 6,6 7,6 15,9 23,4
D1 *** *** * *** *** *** *** * * ***
Dunnet's MC test P > 0.05 P > 0.05 P < 0.01 P > 0.05 P < 0.01 P > 0.05 P < 0.01 P < 0.01 P < 0.01 P < 0.01 P < 0.01 P < 0.05 P < 0.05 P < 0.01
Dunn's MC test P > 0.05 P > 0.05 P < 0.01 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P < 0.01 P < 0.01 P < 0.01 P < 0.01 P > 0.05 P > 0.05 P < 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 147,2 147,6 142,6 144,0 147,4 140,4 136,4 124,6 136,1 137,5 142,1 142,1 139,7 112,9
Median 143,0 147,0 142,0 140,0 147,0 140,0 135,0 122,0 136,0 135,0 140,0 140,0 136,0 111,0
SD 7,1 6,4 2,3 5,3 4,0 1,4 3,0 5,0 4,0 4,1 4,7 6,0 8,3 9,3
D2 *** *** *** * *** * *
Dunnet's MC test P > 0.05 P > 0.05 P < 0.01 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P < 0.05 P < 0.01 P > 0.05 P < 0.05 P < 0.05
Dunn's MC test P > 0.05 P > 0.05 P < 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 149,9 146,5 143,9 145,5 145,9 141,2 139,3 131,4 132,5 141,2 143,8 151,0 140,6 117,2
Median 147,0 140,0 141,0 143,0 142,0 142,0 135,0 133,5 131,0 140,5 144,0 149,5 139,0 116,0
SD 7,5 10,2 5,2 5,6 7,2 6,9 7,6 6,2 3,5 2,7 5,0 12,3 5,1 4,9
D3 * * ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 147,6 142,4 154,4 146,6 168,0 147,1 149,8 151,6 156,3 151,9 155,8 158,9 157,3 125,7
Median 148,5 143,0 154,5 146,0 168,0 150,5 149,5 152,5 157,5 153,5 158,0 160,0 159,5 126,5
SD 5,2 8,3 5,6 6,4 5,9 8,9 5,7 9,7 4,9 10,3 7,3 7,4 9,4 7,2
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)
# no variability in one of the columns

More detailed data are attached as pdf file.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were obtained during ophthalmoscopic examinations.
Individual data are attached as pdf file
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Red Blood Cells Count (RBC, HGB, HCT, MCV, MCH, MCHC), Erythroblasts Count (Ebl)
Usual inter-group variability was observed during the study. The examination performed prior to the administration start (Examination 1) revealed a few statistically significant differences between the control and dosed groups (MCH, MCHC, males), however, these values were within the reference limits1.
In the Examination 2, slight increase of erythrocyte count was observed in all monitored groups as compared to the Examination 1, however without any significant differences between the control and dosed groups. Corresponding changes were observed in HGB and HCT values.
Slightly decreased MCV values were observed in all monitored groups in the Examination 2 as compared to the Examination 1, again, without any statistically significant differences between the control and dosed groups. Corresponded changes were observed in MCH and MCHC.
All mean values varied within the reference limits1.
Normal occurrence of erythroblast (Ebl) was observed in all groups during the study.

White Blood Cells Count and Differential Leucocytes Count (WBC, SN, BN, EO, BA, LY, MO)
Slightly increased mean values of leucocyte count (WBC) were observed in the Examination 2 as compared to the Examination 1 in all monitored groups. In D1 and D2 females, the mean WBC values found in the Examination 2 were statistically significantly lower as compared to the control group, however, it was the most probably due to higher mean WBC value occurred in the control females in the Examination 1 which increased in the Examination 2. All WBC values were within the reference limits1.

No considerable differences between the control and dosed groups were observed in differential of leucocyte count (SN, BN, EO, BA and MO) both in males and females in the course of the study. In the Examination 2, statistically significantly lower mean LY values as compared to the control group were observed in D1 and D2 females (but not in males). This finding can be considered biologically insignificant. All mean values of WBC differential count were within the reference limits1.

Coagulation (PLT, APTT, PT)
Also in coagulation parameters, isolated statistically significant differences between the control and dosed groups were observed both in the Examination 1 (PLT in D2 males, PT in D1, D3 males and D2, D3 females) and 2 (PT, D3 males and females). These findings were incidental only without any biological significance. All mean values of coagulation parameters (PLT, APTT, PT) were within the reference limits1.

Generally, individual and mean values of all monitored parameters varied in some extent during the study, no considerable differences were observed between the dosed and control groups in the end of the administration period.

1. Unpublished data of MediTox s.r.o.: Reference values of hematology 2013-2014, Wistar rat.

Hematology - mean, median and SD values
Tab.: 5
Males
Group Exam. No.: WBC 109/l RBC 1012/l HGB g/l HCT l/l MCV fl MCH pg MCHC g/l PLT 109/l Ebl. ‰ SN 109/l BN 109/l EO 109/l BA 109/l LY 109/l MO 109/l APTT sec. PT sec.
C 1 + + + +
ANOVA p 0,582 0,354 0,903 0,487 0,055 0,020 0,001 0,152 - 0,532 0,161 0,192 - 0,539 0,075 0,300 0,001
K-W test p 0,554 0,232 0,893 0,350 0,101 0,015 0,001 0,040 - 0,397 0,228 0,173 - 0,578 0,048 0,319 0,002
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,73 6,891 147,4 0,4501 65,3 21,40 327,4 826,1 0,0 0,5 0,2 0,066 0,000 6,874 0,070 12,160 16,710
Median 7,45 6,895 147,5 0,4505 65,5 21,45 328,5 819,5 0,0 0,5 0,2 0,071 0,000 6,593 0,034 11,600 16,850
SD 1,03 0,281 5,8 0,0188 1,3 0,64 4,6 88,9 0,0 0,1 0,1 0,070 0,000 0,938 0,099 2,049 0,876
2 + + +
ANOVA p 0,516 0,480 0,887 0,666 0,723 0,492 0,086 0,210 0,404 0,054 0,352 0,256 - 0,487 0,981 0,109 0,005
K-W test p 0,587 0,209 0,900 0,817 0,412 0,104 0,026 0,107 0,392 0,039 0,571 0,214 - 0,469 0,937 0,087 0,009
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 9,23 8,684 159,8 0,4567 52,8 17,48 349,5 797,5 0,0 1,0 0,2 0,106 0,000 7,795 0,138 12,300 14,630
Median 8,90 8,750 162,5 0,4545 51,5 18,05 351,5 782,5 0,0 1,0 0,1 0,108 0,000 7,704 0,103 11,900 14,800
SD 1,39 0,901 9,5 0,0213 3,8 3,60 8,1 110,7 0,0 0,4 0,2 0,065 0,000 1,375 0,096 2,248 0,620
D1 1 *** x *
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P < 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.001 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,61 6,646 146,2 0,4323 65,1 22,01 338,2 793,4 0,0 0,7 0,1 0,053 0,000 6,720 0,048 13,820 15,170
Median 7,80 6,580 146,5 0,4325 65,0 22,15 338,5 795,0 0,0 0,7 0,1 0,032 0,000 6,828 0,000 13,700 15,200
SD 1,19 0,381 7,8 0,0215 1,1 0,38 3,1 132,5 0,0 0,3 0,1 0,065 0,000 1,154 0,072 2,171 0,815
2
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 9,27 8,518 159,5 0,4494 52,7 18,74 355,1 821,2 0,0 1,4 0,1 0,169 0,000 7,366 0,151 14,250 14,840
Median 9,20 8,600 160,0 0,4515 52,0 18,70 356,0 825,5 0,0 1,4 0,1 0,145 0,000 7,354 0,166 14,800 14,800
SD 0,96 0,273 7,2 0,0198 1,1 0,41 2,3 138,8 0,0 0,3 0,1 0,142 0,000 0,998 0,102 1,504 0,337
D2 1 *** **
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P < 0.05 - P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,42 6,561 146,1 0,4384 66,8 22,32 333,2 722,3 0,0 0,7 0,1 0,060 0,000 6,527 0,024 12,830 15,770
Median 7,40 6,600 146,0 0,4410 67,0 22,55 332,5 738,0 0,0 0,6 0,1 0,071 0,000 6,531 0,000 12,100 15,750
SD 0,94 0,379 4,8 0,0175 1,9 0,74 3,5 62,2 0,0 0,3 0,1 0,050 0,000 0,774 0,055 2,582 1,074
2
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 9,61 8,659 158,6 0,4526 52,3 18,32 350,4 698,0 0,0 1,0 0,1 0,212 0,000 8,075 0,155 13,390 14,360
Median 9,65 8,750 159,0 0,4600 52,5 18,30 350,0 683,0 0,0 0,9 0,1 0,211 0,000 7,911 0,181 12,850 14,200
SD 0,96 0,370 6,6 0,0186 1,4 0,39 5,1 136,0 0,0 0,4 0,1 0,130 0,000 0,979 0,113 1,914 0,757
D3 1 *** x ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P < 0.01
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P < 0.01
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 8,13 6,835 148,8 0,4456 65,3 21,80 334,0 813,9 0,0 0,7 0,1 0,015 0,000 7,189 0,125 13,770 14,520
Median 8,65 6,840 147,5 0,4390 65,0 21,65 335,5 811,0 0,0 0,6 0,1 0,000 0,000 7,521 0,092 13,650 14,550
SD 1,45 0,704 14,9 0,0431 1,5 0,70 8,9 129,3 0,0 0,3 0,1 0,033 0,000 1,205 0,107 2,135 1,525
2 x ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P < 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05 P < 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 10,06 8,899 161,2 0,4594 51,8 18,17 351,2 777,5 0,1 1,3 0,2 0,123 0,000 8,213 0,144 14,130 13,760
Median 10,05 8,760 161,5 0,4575 51,5 18,25 350,5 789,0 0,0 1,4 0,1 0,092 0,000 8,464 0,120 13,700 14,050
SD 1,97 0,390 5,7 0,0168 0,9 0,37 2,5 148,3 0,3 0,4 0,2 0,154 0,000 1,700 0,092 1,966 0,810
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)
# no variablity in one of the columns


Tab.: 6
Females
Group Exam. No.: WBC 109/l RBC 1012/l HGB g/l HCT l/l MCV fl MCH pg MCHC g/l PLT 109/l Ebl. ‰ SN 109/l BN 109/l EO 109/l BA 109/l LY 109/l MO 109/l APTT sec.
C 1 + + +
ANOVA p 0,166 0,379 0,710 0,583 0,572 0,651 0,747 0,668 0,404 0,761 0,413 0,504 - 0,149 0,776 0,268
K-W test p 0,166 0,269 0,518 0,416 0,728 0,617 0,659 0,627 0,392 0,857 0,375 0,504 - 0,150 0,794 0,338
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,67 7,316 149,1 0,4343 59,5 20,38 343,1 957,3 0,0 0,5 0,1 0,078 0,000 5,929 0,051 13,740
Median 6,95 7,275 153,0 0,4445 59,0 20,20 342,0 896,0 0,0 0,5 0,1 0,000 0,000 5,956 0,000 13,400
SD 1,75 0,618 12,6 0,0352 1,6 0,75 3,0 171,5 0,0 0,3 0,1 0,123 0,000 1,508 0,077 1,610
2 + + + x + +
ANOVA p 0,009 0,352 0,263 0,296 0,315 0,419 0,425 0,158 0,404 0,903 0,886 0,061 - 0,005 0,266 0,966
K-W test p 0,012 0,467 0,213 0,139 0,396 0,489 0,282 0,198 0,392 0,935 0,992 0,076 - 0,011 0,280 0,460
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,37 7,834 158,0 0,4432 56,5 20,22 356,4 824,6 0,1 0,6 0,1 0,029 0,000 6,617 0,062 14,430
Median 7,55 7,700 159,5 0,4420 56,0 20,25 359,0 864,0 0,0 0,6 0,1 0,000 0,000 6,795 0,062 14,200
SD 1,12 0,509 7,1 0,0157 2,7 0,77 9,6 128,8 0,3 0,2 0,1 0,052 0,000 1,057 0,080 2,085
D1 1
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 8,12 7,466 151,7 0,4415 58,9 20,30 343,3 983,4 0,0 0,7 0,1 0,047 0,000 7,233 0,067 13,920
Median 8,40 7,460 152,0 0,4435 59,5 20,35 344,0 1.027,5 0,0 0,6 0,1 0,000 0,000 7,718 0,025 13,400
SD 1,69 0,227 4,8 0,0127 1,3 0,46 2,8 165,7 0,0 0,3 0,1 0,069 0,000 1,400 0,084 1,717
2 * x * x
Dunnet's MC test P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P < 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,11 7,817 154,9 0,4753 55,2 19,86 360,2 786,2 0,0 0,6 0,1 0,085 0,000 5,380 0,012 13,620
Median 6,15 7,840 155,5 0,4365 55,5 19,95 359,5 780,5 0,0 0,5 0,1 0,070 0,000 5,406 0,000 11,950
SD 1,00 0,486 9,2 0,1262 1,6 0,65 4,2 104,6 0,0 0,3 0,0 0,077 0,000 0,740 0,026 5,357
D2 1
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,66 7,607 152,8 0,4469 58,7 20,11 342,0 930,6 0,0 0,6 0,1 0,068 0,000 6,870 0,047 12,660
Median 7,40 7,715 153,0 0,4480 59,0 20,15 341,5 954,5 0,0 0,5 0,1 0,069 0,000 6,828 0,035 12,650
SD 1,43 0,376 6,8 0,0181 1,3 0,45 2,6 215,4 0,0 0,2 0,1 0,070 0,000 1,290 0,051 1,533
2 *** ***
Dunnet's MC test P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P < 0.01 P > 0.05 P > 0.05
Dunn's MC test P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P < 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 5,98 7,802 154,6 0,4308 55,4 19,81 358,7 745,4 0,0 0,6 0,1 0,090 0,000 5,162 0,041 14,190
Median 5,85 7,870 158,5 0,4425 55,0 19,65 359,0 757,5 0,0 0,7 0,1 0,066 0,000 5,180 0,028 14,600
SD 0,44 0,412 9,1 0,0247 1,2 0,50 2,4 55,5 0,0 0,2 0,1 0,044 0,000 0,379 0,048 3,583
D3 1 x
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,12 7,356 149,7 0,4362 59,2 20,37 342,8 1.028,3 0,1 0,6 0,1 0,025 0,000 6,392 0,076 13,810
Median 7,15 7,305 149,0 0,4345 59,0 20,45 341,5 1.018,0 0,0 0,6 0,1 0,000 0,000 6,469 0,072 13,700
SD 0,96 0,257 5,3 0,0150 1,1 0,39 2,9 169,8 0,3 0,2 0,1 0,053 0,000 0,961 0,066 1,469
2 x
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 - P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,12 7,502 150,4 0,4215 56,3 20,07 357,0 844,7 0,0 0,6 0,1 0,121 0,000 6,252 0,039 14,240
Median 7,15 7,680 152,5 0,4295 56,0 19,85 358,0 822,5 0,0 0,7 0,1 0,118 0,000 6,117 0,000 13,850
SD 1,38 0,475 7,9 0,0210 1,5 0,49 2,9 106,3 0,0 0,3 0,1 0,107 0,000 1,383 0,055 2,989
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)
# no variablity in one of the columns

More detailed data are attached as pdf file.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The individual and mean values of the most serum chemistry parameters varied within the reference limits1 during the study. In isolated cases (various groups), several mean values of some serum chemistry parameters slightly exceeded the reference limits either in the Examination 1 or Examination 2 (LDH, ALP and ALT in males and P, Crea, ALT, ALP, Chol, TP and Glo in females).
As far as the course of the study is concerned, the individual and mean values varied slightly in both examinations in all the monitored groups, only a few statistically significant differences were observed in dosed groups as compared to the control group. The statistical differences found were either present in the Examination 1 (before the start of the administration) or they were found in one sex only. Moreover, there was not proved any dependence on the dose administered. Hence, these findings were considered biologically insignificant.
Generally, normal variations were observed in Glu, Na, K, Cl, Ca serum concentrations during the study.
Slightly more pronounced inter-group variations were observed in P serum concentration in males and females, however, without any substantial differences between control and dosed groups.
Mean values of urea, creatinine, bile acids and bilirubin serum concentrations varied mostly within the reference limits. Slight increase of creatinine serum concentration was observed in all the groups in the Examination 2 as compared to the Examination 1, no statistically significant differences were observed between the control and dosed groups.
More pronounced inter-individual and inter-group variations were observed in LDH serum activity, however all the mean values varied within the reference limits1.
Slight increased ALT serum activity was observed in all the groups both in males and females in the Examination 2 as compared to the Examination 1 even up to the upper reference limit. No substantial differences were observed between the control and dosed groups. On the other hand, AST mean values varied within the reference limits in all the groups (males and females) both in the Examination 1 and 2.
Statistically significant increase of GMT mean values was identified in D3 males and females in the Examination 2 as compared to the control group. While in the other groups (C, D1 and D2) mean GMT serum activity slightly decreased in the Examination 2 as compared to the Examination 1 (15 – 20 % in average), in D3 males and females this value increased by 10 % in average. However, the upper reference limit was not exceeded.
Mean values of ALP serum activity decreased in all the monitored groups in the Examination 2 as compared to the Examination 1 even below to lower reference limit, however, at the end of the administration period the animals were 3 months older than those the reference limits are calculated for. It is well known that ALP values decrease with advancing age of the animals.
Chol and TGC mean values revealed also more pronounced inter-group variations with some statistically significant differences identified between the control and dosed groups, however, no clear dose dependence was proved and the reference limits were not exceeded.
Slight increase of total protein serum concentration was observed both in males and females of all monitored group in the Examination 2 as compared to the Examination 1. In D3 females this increase was also statistically significant as compared to the control group and exceeding the upper reference limit. While in the other groups the increase in the Examination 2 (as compared to the Examination 1) varied within 15 – 17 %, in D3 females it was by 31%. Similar and statistically significant increase in globulin serum concentration was observed in D3 females in the Examination 2 as compared to the control group. This increase in total protein was a consequence of a slight increase in globulin serum concentrations in all the groups in the Examination 2 as compared to the Examination 1 (15 – 20 % in average) while in D3 females it was by 34 %.

These changes resulted in statistically significantly decreased Alb/Glo ratio in D3 females in the Examination 2 as compared to the control group.
No substantial changes were observed in albumin serum concentration.
1. Unpublished data of MediTox s.r.o.: Reference values of serum chemistry 2013-2014, Wistar rat.

Clinical Chemistry - mean, median and SD values
Tab.: 5
Males
Group Exam. No.: GLU mmol/l Na mmol/l K mmol/l Cl mmol/l Ca mmol/l P mmol/l Urea mmol/l Crea mmol/l Bil µmol/l BA mmol/l LDH µkat/l ALT µkat/l AST µkat/l GMT µkat/l ALP µkat/l Chol mmol/l TGC mmol/l TP g/l Alb g/l Glo g/l Alb/Glo ratio
C 1 x+ +
ANOVA p P<0.0001 0,097 0,946 0,319 0,005 0,003 0,022 0,036 0,568 0,141 0,117 0,979 0,644 0,206 0,374 0,003 0,001 0,896 0,714 0,961 0,874
K-W test p 0,001 0,111 0,985 0,332 0,006 0,009 0,019 0,041 0,317 0,075 0,093 0,683 0,597 0,208 0,220 0,004 0,001 0,917 0,775 0,937 0,809
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,587 140,0 4,37 96,7 2,668 2,691 8,33 20,8 2,7 78,9 15,414 0,911 1,702 0,142 4,457 1,858 0,779 53,3 11,8 41,5 0,285
Median 6,495 139,0 4,35 96,0 2,680 2,645 8,00 20,0 3,0 56,5 16,370 0,860 1,845 0,150 4,275 1,855 0,710 53,0 12,0 41,5 0,284
SD 0,690 2,7 0,40 3,0 0,119 0,218 1,30 4,8 0,5 52,4 4,510 0,199 0,264 0,029 0,793 0,156 0,249 2,9 0,6 2,5 0,015
2 + x + +
ANOVA p 0,749 0,003 0,124 0,054 0,002 0,168 0,126 0,435 0,269 0,379 0,001 0,102 0,218 P<0.0001 0,007 0,815 0,057 0,325 0,891 0,142 0,126
K-W test p 0,729 0,005 0,133 0,143 0,008 0,150 0,197 0,666 0,205 0,478 0,005 0,199 0,204 0,000 0,015 0,828 0,018 0,193 0,890 0,086 0,146
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,456 140,6 4,35 100,4 2,457 2,292 6,39 30,9 2,7 52,8 11,539 1,055 1,656 0,115 1,788 1,847 0,922 61,2 12,3 48,9 0,252
Median 7,375 141,0 4,40 100,5 2,470 2,235 6,35 31,0 3,0 55,0 11,945 0,980 1,665 0,115 1,875 1,900 0,785 61,0 12,0 49,0 0,250
SD 0,836 2,0 0,24 1,4 0,070 0,125 0,62 4,2 0,9 25,8 2,550 0,203 0,343 0,018 0,230 0,178 0,330 2,3 0,7 1,8 0,011
D1 1 * *** ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,007 139,4 4,32 97,6 2,690 2,944 7,50 19,6 2,2 51,7 11,795 0,917 1,569 0,135 4,750 1,629 1,585 53,1 11,7 41,4 0,282
Median 7,120 139,5 4,30 98,0 2,705 2,940 7,30 20,0 2,0 41,5 11,370 0,930 1,510 0,140 4,500 1,640 1,660 54,0 12,0 42,0 0,282
SD 0,534 2,5 0,30 2,2 0,088 0,195 0,95 3,4 0,9 33,2 3,091 0,126 0,192 0,013 0,900 0,173 0,733 2,6 0,8 1,8 0,012
2 * ***
Dunnet's MC test P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,389 143,6 4,11 101,2 2,488 2,279 6,46 33,1 2,0 40,9 8,261 1,092 1,609 0,120 1,422 1,847 0,943 60,4 12,6 47,8 0,263
Median 7,370 143,5 4,10 101,5 2,475 2,290 6,90 32,5 2,0 39,0 8,055 1,120 1,515 0,120 1,390 1,870 0,835 59,5 12,5 47,0 0,262
SD 0,510 2,3 0,21 2,3 0,060 0,183 1,12 5,6 1,2 20,6 2,499 0,209 0,346 0,009 0,105 0,223 0,421 2,4 1,0 1,7 0,016
D2 1 *** *** *** x *** ***
Dunnet's MC test P < 0.01 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P < 0.01 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,583 141,4 4,31 98,6 2,814 3,015 7,71 22,7 2,3 100,9 11,636 0,944 1,635 0,123 4,992 1,615 1,364 53,8 12,0 41,8 0,287
Median 7,470 142,0 4,30 99,0 2,810 3,030 7,85 24,0 2,0 104,0 10,095 0,905 1,555 0,125 4,970 1,660 1,290 53,5 12,0 41,5 0,291
SD 0,475 1,8 0,16 2,2 0,076 0,119 1,06 3,2 0,5 53,0 4,389 0,159 0,363 0,016 0,620 0,159 0,481 2,3 0,7 1,9 0,013
2 *
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,174 142,6 4,31 100,6 2,554 2,427 7,25 34,1 2,4 43,8 14,990 1,093 1,928 0,125 1,536 1,945 0,851 62,3 12,5 49,8 0,251
Median 6,995 143,0 4,35 101,0 2,580 2,495 7,10 33,0 3,0 40,0 15,900 1,015 1,940 0,120 1,510 1,935 0,895 61,5 12,5 49,5 0,247
SD 0,850 1,6 0,22 1,7 0,113 0,204 0,89 6,8 1,0 25,4 4,893 0,321 0,410 0,013 0,276 0,356 0,231 3,9 0,8 3,3 0,016
D3 1
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,357 139,0 4,37 97,1 2,667 2,887 8,89 16,4 2,3 87,8 14,457 0,910 1,563 0,134 4,426 1,825 0,779 53,8 12,0 41,8 0,288
Median 6,370 139,0 4,35 97,5 2,655 2,870 8,80 16,5 3,0 68,5 13,965 0,775 1,530 0,135 4,260 1,835 0,705 54,0 12,0 42,0 0,284
SD 0,481 1,7 0,23 1,9 0,104 0,198 0,79 6,6 1,3 47,8 4,423 0,288 0,256 0,017 0,918 0,197 0,259 2,1 0,7 2,0 0,021
2 *** ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,197 139,2 4,27 98,5 2,392 2,364 6,79 30,9 2,8 34,9 10,030 1,454 1,931 0,148 1,395 1,924 0,594 62,7 12,5 50,2 0,249
Median 7,170 140,0 4,25 99,0 2,395 2,355 6,95 31,0 3,0 37,5 9,995 1,255 1,805 0,150 1,350 1,795 0,530 63,5 13,0 50,5 0,250
SD 0,490 4,0 0,26 3,0 0,099 0,124 0,77 3,9 0,8 19,1 3,264 0,668 0,602 0,011 0,361 0,355 0,189 3,3 1,0 2,5 0,013
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)
# no variablity in one of the columns


Tab.: 6
Females
Group Exam. No.: GLU mmol/l Na mmol/l K mmol/l Cl mmol/l Ca mmol/l P mmol/l Urea mmol/l Crea mmol/l Bil µmol/l BA mmol/l LDH µkat/l ALT µkat/l AST µkat/l GMT µkat/l ALP µkat/l Chol mmol/l TGC mmol/l TP g/l Alb g/l Glo g/l Alb/Glo ratio
C 1 + +
ANOVA p 0,006 0,095 0,343 0,769 0,015 0,738 0,076 0,670 0,517 0,957 0,366 0,001 0,787 0,309 0,088 0,173 0,009 0,344 0,539 0,385 0,944
K-W test p 0,009 0,042 0,525 0,748 0,025 0,809 0,062 0,723 0,399 0,739 0,485 0,003 0,729 0,452 0,197 0,185 0,019 0,381 0,592 0,464 0,842
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,464 141,1 3,76 99,8 2,607 2,530 6,98 20,6 2,1 53,1 12,234 0,632 1,490 0,135 2,728 2,141 0,547 57,0 12,8 44,2 0,290
Median 6,245 141,0 3,70 100,0 2,600 2,505 6,30 21,0 2,5 26,0 12,630 0,645 1,445 0,135 2,605 2,140 0,520 57,5 13,0 44,0 0,289
SD 0,791 1,4 0,18 1,7 0,094 0,241 1,80 3,6 1,2 61,3 3,512 0,178 0,187 0,014 0,709 0,348 0,149 2,7 0,8 2,3 0,017
2 + + x + + + + +
ANOVA p 0,283 0,006 0,879 0,179 0,306 0,007 0,002 0,614 0,021 0,021 0,521 0,202 0,731 0,008 0,002 P<0.0001 0,005 0,002 0,901 P<0.0001 P<0.0001
K-W test p 0,819 0,008 0,729 0,206 0,128 0,020 0,004 0,722 0,003 0,001 0,089 0,267 0,828 0,036 0,001 P<0.0001 0,023 0,001 0,970 0,000 0,001
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,862 142,0 3,65 102,0 2,461 1,930 7,15 39,0 2,6 69,5 9,906 1,085 1,537 0,129 1,247 1,774 0,653 66,2 14,8 51,4 0,288
Median 6,830 142,5 3,60 101,5 2,455 1,945 7,10 37,0 3,0 54,0 9,040 1,065 1,430 0,125 1,130 1,940 0,585 67,0 15,0 52,0 0,290
SD 0,417 1,6 0,21 2,4 0,086 0,172 1,11 5,5 1,0 51,8 4,322 0,418 0,306 0,014 0,602 0,711 0,240 4,3 1,1 3,6 0,019
D1 1
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,881 139,2 3,80 99,2 2,630 2,486 6,69 19,1 2,2 53,9 11,325 0,671 1,494 0,145 2,601 1,943 0,597 55,6 12,6 43,0 0,293
Median 6,835 139,5 3,80 99,0 2,610 2,495 6,85 19,5 2,0 21,5 11,095 0,605 1,435 0,140 2,510 1,920 0,525 56,0 12,5 43,0 0,294
SD 0,721 2,1 0,18 2,0 0,075 0,191 1,52 2,8 1,0 56,8 2,234 0,158 0,296 0,018 0,350 0,328 0,188 1,7 0,7 1,3 0,015
2 ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,751 143,6 3,72 102,8 2,529 2,097 6,62 37,5 2,7 63,3 11,863 0,965 1,625 0,122 1,072 1,819 1,063 65,7 15,1 50,6 0,298
Median 6,750 144,0 3,70 103,5 2,520 2,100 6,60 38,5 3,0 55,0 12,515 0,880 1,500 0,125 1,065 1,795 1,100 65,0 14,5 50,5 0,296
SD 0,483 1,3 0,20 1,8 0,063 0,185 0,63 5,3 0,7 49,8 2,190 0,374 0,528 0,014 0,288 0,229 0,385 5,9 1,7 4,3 0,015
D2 1 *
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,117 140,0 3,90 99,0 2,702 2,449 7,27 18,4 1,5 52,5 12,768 0,796 1,498 0,132 3,224 2,273 0,616 56,5 12,8 43,7 0,293
Median 7,195 141,0 3,90 99,5 2,715 2,395 7,65 20,0 2,0 44,5 11,040 0,815 1,425 0,130 3,020 2,300 0,570 56,5 13,0 44,0 0,294
SD 0,378 3,0 0,28 3,2 0,086 0,173 1,37 7,7 1,1 30,2 3,886 0,107 0,185 0,018 1,059 0,369 0,158 2,6 0,8 1,9 0,010
2 x**
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 7,481 141,5 3,64 101,2 2,510 1,839 7,78 39,2 1,6 71,8 11,375 0,927 1,509 0,132 1,148 1,986 0,643 64,1 14,7 49,4 0,298
Median 7,025 141,5 3,50 101,0 2,520 1,825 8,35 38,5 2,0 64,0 9,010 0,910 1,400 0,130 1,015 1,960 0,590 67,0 15,0 51,5 0,300
SD 1,593 2,1 0,45 1,8 0,117 0,167 1,25 5,2 0,8 40,5 5,653 0,143 0,337 0,013 0,439 0,275 0,197 5,4 1,4 4,1 0,015
D3 1 ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,000 138,3 3,74 98,7 2,559 2,536 5,56 17,5 2,0 64,7 10,458 0,532 1,413 0,132 2,472 2,007 0,828 55,2 12,4 42,8 0,290
Median 5,705 139,0 3,75 98,5 2,570 2,465 5,25 20,0 2,0 49,5 10,050 0,535 1,365 0,135 2,485 2,070 0,775 55,5 12,0 43,0 0,287
SD 0,846 3,0 0,17 2,3 0,116 0,181 1,25 7,1 1,2 70,7 2,368 0,076 0,174 0,020 0,303 0,355 0,236 2,5 0,5 2,3 0,016
2 * *** * *** *** *** *** ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P < 0.01 P < 0.01 P > 0.05 P < 0.05 P > 0.05 P < 0.01 P < 0.01
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P < 0.01 P > 0.05 P < 0.05 P > 0.05 P < 0.05 P < 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Mean 6,857 140,7 3,62 101,0 2,531 1,794 5,85 36,5 2,8 18,4 9,320 0,792 1,437 0,147 0,543 2,652 0,727 72,3 15,0 57,3 0,262
Median 6,940 140,5 3,60 101,0 2,530 1,745 5,75 36,5 3,0 14,0 8,445 0,790 1,440 0,150 0,470 2,630 0,655 72,5 15,0 57,5 0,261
SD 0,642 1,9 0,22 1,9 0,094 0,245 1,13 4,7 1,1 10,8 4,444 0,156 0,271 0,020 0,199 0,206 0,264 1,9 0,8 1,6 0,015
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)
# no variablity in one of the columns

More detailed data are attached as pdf file.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urine samples of the tested and control animals were mostly clear and of yellow colour during the entire study except of slightly turbid samples found in some of males in the control and D3 groups in Examination 2.

No substantial differences were observed between the dosed and control groups in the volume of urine samples collected.

Normal findings were observed in glucose, bilirubin and nitrites occurrence in all monitored groups in the course of the study.
pH values varied mostly within 6.0 – 8.5 with sporadic values of 5.0 and 9.0. Specific gravity varied mostly within <1.005 – 1.015 kg/l, sometimes the value >1.030 kg/l was observed independently on the dose or the examination.

Increased protein occurrence was observed in most of males of all monitored groups in the Examination 2. Increased values varied within 0.5 – 3.0 g/l (9 males from 10) and >6.0 g/l in one case in control males while in D3 males increased values varied within 1.0 - >6.0 (10 males from 10). In control females, only isolated slightly higher values were observed in the Examination 2 (0.2 – 0.5 g/l in 2 females from 10). In D1 and D2 females protein occurrence varied both in the Examination 1 and 2 and in D3 females slightly increased values were observed (0.5 – 1.0 g/l, 6.0 g/l in one case) in 8 females from 10 in the Examination 2.

Slightly increased urobilinogen occurrence was observed in some animals of all the groups in the Examination 1.

More variations were observed in blood occurrence in urine samples between the groups, some increased values were observed in all the groups in both Examinations (mainly in males of control and D3 groups in Examination 2). No considerable differences were found between the control and dosed groups.

Slight to moderate presence of leucocytes (25 – 500 Leu/µl) was observed almost in all males and in most of females throughout the study independently on the dose and examination. No substantial difference was found between the dosed and control groups.

Higher variability of ketones occurrence was observed in most of animals in all monitored groups in the Examination 1 (1.0 – 4.0 mmol/l). In the Examination 2, increased ketones occurrence was observed in control males (4 mmol/l, 3 males from 10), while in control females ketones occurrence decreased mostly. In D1 and D2 animals, ketones occurrence in the Examination 2 either remained as in the Examination 1 or decreased. On the contrary, in D3 males ketones occurrence increased in the Examination 2 to 4 – 6 mmol/l in 8 males from 10 while in females, it either decreased to normal values or it remained as in the Examination 1.

A detailed overview is attached as pdf file.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males:
There was found significant increase of relative liver weight in D1 males and of absolute and relative liver weight in D2 and D3 males. The relative weight of kidneys was higher in D2 males and absolute and relative kidney weight was increased in D3 males. Significant decrease of absolute weight of thyroid gland was found in D2 males. In D3 males increased relative weights of the brain, adrenals and testes were noted.

Females:
Relative and absolute weights of the liver and kidneys were increased in D3 females. Significant decrease of absolute and relative weight of thyroid gland was found in D1 females.

Significant increase of absolute and relative liver weight found in D3 males and females and in D1 and D2 males was probably related to the Test Item administered despite of there were no considerable changes in histopathology examination of this organ.
Increase of relative kidneys weight in D2 and D3 males and D3 females was most probably related to the Test item administered and except D3 females it is in correlation with changes observed in histopathology examination.

Summary:
1000 mg/kg bw: M+ F significantly increased kidney weight abs/rel; F significantly increased liver weight abs/rel; M significantly increased brain, adrenals and testes weight rel
300 and 1000 mg/kg bw: M significantly increased liver weight abs/rel (DR)
300 mg/kg bw: M significantly increased kidney weight rel



ABSOLUTE ORGAN WEIGHT (g) - mean, median and SD values
Tab.: 5
Males
Group Exam. No.: Body weight Liver Kidneys Spleen Brain Heart Thymus Adrenals Thyroid gland Testes Uterus Prostate gl.Ovaries Epididym.
C +
ANOVA p 0,003 0,000 0,026 0,692 0,721 0,064 0,431 0,122 0,005 0,092 0,938 0,483
K-W test p 0,008 0,002 0,030 0,682 0,333 0,035 0,259 0,121 0,015 0,290 0,982 0,413
N 10 10 10 10 10 10 10 10 10 10 10 10
Mean 550,10 16,147 3,642 0,958 1,986 1,708 0,354 0,0826 0,0345 4,1400 0,9383 1,7028
Median 562,00 16,190 3,610 0,965 1,980 1,691 0,338 0,0823 0,0346 4,1600 0,9085 1,6390
SD 33,55 1,294 0,201 0,171 0,092 0,204 0,074 0,0178 0,0052 0,3217 0,2587 0,1778
D1
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10
Mean 524,30 16,990 3,875 0,969 2,037 1,712 0,323 0,0826 0,0288 4,1470 0,9609 1,7255
Median 519,00 16,360 3,900 0,968 2,036 1,616 0,293 0,0861 0,0287 4,1400 0,9375 1,7005
SD 45,33 1,991 0,553 0,196 0,107 0,298 0,088 0,0092 0,0057 0,1965 0,2182 0,1196
D2 *** ***
Dunnet's MC test P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10
Mean 509,10 18,614 4,146 0,981 2,022 1,549 0,351 0,0782 0,0270 4,1160 1,0092 1,7081
Median 514,50 18,910 4,135 0,974 2,012 1,513 0,337 0,0750 0,0273 4,0100 0,9345 1,6660
SD 36,23 1,344 0,558 0,140 0,088 0,216 0,072 0,0089 0,0028 0,2333 0,3368 0,2057
D3 *** *** ***
Dunnet's MC test P < 0.01 P < 0.01 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P < 0.01 P < 0.01 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 10
Mean 479,00 19,698 4,356 0,904 2,032 1,475 0,304 0,0918 0,0342 4,4360 0,9538 1,6165
Median 481,00 19,860 4,195 0,883 2,063 1,444 0,298 0,0890 0,0349 4,3100 0,9135 1,5920
SD 40,88 2,377 0,680 0,085 0,138 0,188 0,078 0,0127 0,0068 0,4461 0,2169 0,1600
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)

Tab.: 6
Females
Group Exam. No.: Body weight Liver Kidneys Spleen Brain Heart Thymus Adrenals Thyroid gland Testes Uterus Prostate gl.Ovaries Epididym.
C +
ANOVA p 0,075 P<0.0001 P<0.0001 0,680 0,965 0,838 0,274 0,067 0,021 0,176 0,501
K-W test p 0,102 P<0.0001 0,005 0,778 0,950 0,903 0,361 0,112 0,029 0,133 0,477
N 10 10 10 10 10 10 10 10 10 10 10 -
Mean 317,90 10,772 2,181 0,677 1,863 1,069 0,329 0,1175 0,0291 1,0484 0,2168 -
Median 319,50 10,495 2,201 0,663 1,870 1,060 0,289 0,1138 0,0298 1,0055 0,2288 -
SD 27,06 1,101 0,232 0,128 0,117 0,104 0,097 0,0177 0,0051 0,3366 0,0550 -
D1 ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 -
Mean 315,70 11,241 2,141 0,725 1,846 1,073 0,283 0,1072 0,0227 0,7835 0,1813 -
Median 319,00 10,905 2,141 0,703 1,831 1,087 0,270 0,1011 0,0224 0,6950 0,1752 -
SD 17,59 1,239 0,186 0,126 0,114 0,056 0,063 0,0221 0,0033 0,1765 0,0419 -
D2
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 -
Mean 317,40 11,666 2,151 0,736 1,865 1,071 0,288 0,1201 0,0266 0,8988 0,1855 -
Median 316,50 11,445 2,138 0,726 1,897 1,064 0,286 0,1192 0,0261 0,8995 0,1793 -
SD 15,05 0,864 0,136 0,092 0,133 0,147 0,062 0,0177 0,0055 0,2704 0,0515 -
D3 *** ***
Dunnet's MC test P > 0.05 P < 0.01 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P < 0.01 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10 -
Mean 338,60 15,088 2,585 0,713 1,842 1,112 0,335 0,1294 0,0264 0,8769 0,1969 -
Median 331,00 15,475 2,612 0,675 1,868 1,102 0,325 0,1335 0,0263 0,7830 0,1782 -
SD 24,74 1,505 0,294 0,101 0,128 0,156 0,066 0,0133 0,0030 0,2437 0,0717 -
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)


ORGAN-TO BODY WEIGHT RATIO (g/kg) - mean, median and SD values
Tab.: 5
Males
Group Exam. No.: Liver Kidneys Spleen Brain Heart Thymus Adrenals Thyroid gland Testes Uterus Prostate gl.Ovaries Epididym.
C + + +
ANOVA p P<0.0001 P<0.0001 0,352 0,002 0,611 0,639 0,001 0,003 0,000 0,484 0,294
K-W test p P<0.0001 P<0.0001 0,640 0,011 0,696 0,442 0,005 0,005 0,004 0,519 0,360
N 10 10 10 10 10 10 10 10 10 10 10
Mean 29,38 6,633 1,741 3,621 3,102 0,642 0,150 0,0630 7,556 1,6975 3,1046
Median 29,38 6,503 1,824 3,535 3,093 0,643 0,146 0,0627 7,409 1,6313 3,1278
SD 1,93 0,388 0,298 0,258 0,286 0,115 0,031 0,0107 0,798 0,4169 0,3581
D1 *
Dunnet's MC test P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10
Mean 32,39 7,395 1,841 3,900 3,270 0,613 0,158 0,0555 7,943 1,8301 3,3191
Median 32,77 7,410 1,876 3,896 3,118 0,583 0,160 0,0501 7,937 1,7070 3,2614
SD 2,10 0,903 0,290 0,251 0,536 0,152 0,021 0,0133 0,508 0,3781 0,4156
D2 *** ***
Dunnet's MC test P < 0.01 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P < 0.01 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10
Mean 36,61 8,127 1,922 3,986 3,039 0,684 0,154 0,0531 8,117 2,0040 3,3605
Median 36,80 7,961 1,913 3,907 2,976 0,674 0,147 0,0547 8,317 1,8799 3,2577
SD 1,88 0,731 0,187 0,287 0,333 0,099 0,016 0,0049 0,651 0,7072 0,3744
D3 *** *** *** *** ***
Dunnet's MC test P < 0.01 P < 0.01 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P < 0.01 P > 0.05 P > 0.05
Dunn's MC test P < 0.01 P < 0.01 P > 0.05 P < 0.01 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 10
Mean 41,08 9,092 1,889 4,275 3,091 0,631 0,192 0,0715 9,326 2,0075 3,3785
Median 41,90 8,920 1,907 4,384 2,975 0,640 0,198 0,0707 9,409 1,9693 3,3784
SD 3,03 1,140 0,111 0,522 0,422 0,129 0,025 0,0135 1,219 0,5089 0,2393
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)

Tab.: 6
Females
Group Exam. No.: Liver Kidneys Spleen Brain Heart Thymus Adrenals Thyroid gland Testes Uterus Prostate gl.Ovaries Epididym.
C
ANOVA p P<0.0001 0,000 0,288 0,374 0,813 0,423 0,225 0,018 0,209 0,424
K-W test p P<0.0001 0,003 0,297 0,347 0,633 0,499 0,315 0,050 0,258 0,259
N 10 10 10 10 10 10 10 10 10 10 -
Mean 33,95 6,866 2,126 5,905 3,371 1,030 0,370 0,0918 3,333 0,6823 -
Median 33,11 6,741 2,129 5,728 3,393 0,899 0,366 0,0873 3,063 0,7422 -
SD 2,87 0,525 0,345 0,690 0,301 0,259 0,051 0,0150 1,160 0,1653 -
D1 ***
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.01 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 -
Mean 35,59 6,781 2,296 5,866 3,404 0,894 0,338 0,0723 2,503 0,5724 -
Median 34,44 6,724 2,223 5,764 3,423 0,866 0,322 0,0731 2,179 0,5511 -
SD 3,13 0,414 0,366 0,535 0,183 0,183 0,060 0,0120 0,653 0,1198 -
D2
Dunnet's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 -
Mean 36,73 6,778 2,320 5,891 3,370 0,911 0,379 0,0837 2,845 0,5837 -
Median 36,34 6,647 2,312 5,965 3,245 0,891 0,381 0,0828 2,721 0,5449 -
SD 1,34 0,345 0,288 0,547 0,393 0,205 0,059 0,0158 0,894 0,1610 -
D3 *** ***
Dunnet's MC test P < 0.01 P < 0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
Dunn's MC test P < 0.01 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
N 10 10 10 10 10 10 10 10 10 10 -
Mean 44,57 7,619 2,104 5,480 3,277 0,986 0,383 0,0782 2,629 0,5820 -
Median 44,56 7,724 2,038 5,367 3,361 0,978 0,396 0,0773 2,266 0,5491 -
SD 3,26 0,443 0,223 0,706 0,310 0,167 0,035 0,0100 0,892 0,2100 -
Statistically significant difference at the 95.0 % confidence level is pointed up in bold
x Normality test not passed, which tends to invalidate the tests comparing the standard deviations
* Statistically significant difference only between means test groups D1, D2 or D3 versus control group C
** Statistically significant difference only between medians test groups D1, D2 or D3 versus control group C
*** Statistically significant difference between means and medians test groups D1, D2 or D3 versus control group C
+ statisticaly significant difference among standard deviations (F test)


More detailed individual information are attached as pdf file.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Liver
The processus papillaris of the liver in one D3 female (F73) was macroscopically of whitish colour.
There were no pathological findings in the liver related to the treatment.

Kidneys
The kidneys of two D3 males were mildly enlarged and of other four males from this group they were of pale colour.
Mildly enlarged kidneys were also recorded in five D2 males and pale colour in six males. In the D1 males the kidneys were enlarged in three animals with pale colour in four cases.
The described findings were not found in the kidneys of females of all administered groups despite the kidneys of two D3 females were mildly enlarged and in three cases they were of pale colour.

Other lesions in the kidneys concerned either control rats only (medullary cyst) or both control and administered animals (hydronephrosis), therefore they were not related to the Test items administered. Mild corticomedullary mineralization found in the kidneys of one D3 female and chronic pyelitis revealed in two D3 females were of spontaneous origin.

Lungs
Small foci of lipidosis were observed in the lungs of seven males and one female of the D3 group and three males and one female of the control group. Increased frequency of this finding in D3 males led to subsequent examination of the lungs of animals from the middle and low dose groups which showed presence of this lesion in one D1 male and two D2 males. Accordingly, a relation to treatment for this finding cannot be excluded for D3 males.

Small foci of chronic inflammation revealed in the lungs of two D3 males were most probably of spontaneous origin as well as a small osteoma racemosum observed in the lungs of one D2 male.
Blood aspiration, focal hemorrhage, acute emphysema and edema found in the lungs of some control and administered rats were terminal lesions without direct relation to the substances tested.

Gastrointestinal tract
No treatment-related findings were revealed in organs of the gastrointestinal tract.




Short summary:
enlarged kidney: M control 0/10,100 mg/kg bw 3/10, 300 mg/kg bw 5/10, 1000 mg/kg bw 2/10; F 1000 mg/kg bw 2/10
pale kidney: M control 0/10,100 mg/kg bw 4/10, 300 mg/kg bw 6/10, 1000 mg/kg bw 4/10; F 1000 mg/kg bw 3/10

Tab. 17: Gross pathology findings

Dose group: C D1 D2 D3
Sex : M F M F M F M F
Number of animals: 10 10 10 10 10 10 10 10
Organ findings:
Kidneys
- enlarged - - 3 - 5 - 2 2
- hydronephrosis - - 1 - - - - -
- pale colour - - 4 - 6 - 4 3
Liver
proc.papillaris
- whitish capsule - - - - - - - 1
Lungs
- blood aspiration - - 1 1 - 2 - -
- focal hemorrhage 1 - - - 3 1 2 -
Ovary
- cyst NA - NA - NA - NA 1
Lymph nodes
Iliac
- hemorrhage - - - - 1 - 1 1
- greenish colour - - - - - - 1 -
Renal
- hemorrhage - - - - 2 - - -
Thymus
- focal hemorrhage 1 1 2 - 2 - 2 1
Uterus
- dilatation NA 7 NA 2 NA 3 NA 5



More detailed and individual information are attached as pdf file.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver
Histopathological examination revealed hemorrhagic infarsation of this lobe caused most probably by its torsion. This lesion is not in direct relation to the Test item administered.

Kidneys
Histopathological examination revealed in all D3 males marked hyaline droplet formation in the renal tubules together with focal tubular basophilia and necrosis of some tubular epithelial cells and presence of hyaline casts. The droplets contain α2µ globulin and its deposition in renal tubules of males of all dose groups found by immunohistochemistry examination corresponds with our findings (see appendix of the final report). Although small amount of hyaline droplets was found also in control males as a spontaneous finding, this lesion is directly caused by the Test item administered.
Histopathological findings in D1 and D2 groups were of the same character as in the D3 males, however with a milder intensity in the D1 males.

Other lesions in the kidneys concerned either control rats only (medullary cyst) or both control and administered animals (hydronephrosis), therefore they were not related to the Test items administered. Mild corticomedullary mineralization found in the kidneys of one D3 female and chronic pyelitis revealed in two D3 females were of spontaneous origin.

Lungs
Small foci of lipidosis were observed in the lungs of seven males and one female of the D3 group and three males and one female of the control group. Increased frequency of this finding in D3 males led to subsequent examination of the lungs of animals from the middle and low dose groups which showed presence of this lesion in one D1 male and two D2 males. Accordingly, a relation to treatment for this finding cannot be excluded for D3 males.

Small foci of chronic inflammation revealed in the lungs of two D3 males were most probably of spontaneous origin as well as a small osteoma racemosum observed in the lungs of one D2 male.
Blood aspiration, focal hemorrhage, acute emphysema and edema found in the lungs of some control and administered rats were terminal lesions without direct relation to the substances tested.

Hematopoietic and lymphatic systems
Cervical lymph nodes of some control and administered animals showed signs of mild to moderate reactive hyperplasia. Origin of this finding is not clear but its relation to the Test item administered could be excluded.
Mild hemorrhage revealed in the cervical lymph nodes of three administered males is a terminal lesion without direct relation to the treatment.
Mild hemorrhage in the iliac lymph nodes was grossly found in one male and one female from the D3 dose group. Histopathological examination confirmed this finding in the male together with presence of hemosiderin. Relation of this findings to the Test item administered is not clear.

Microscopic examination of the spleen did not reveal any pathological changes except for marked venostasis observed in six D3 males as a terminal finding not directly related to the Test item.

Small cysts found in the thymus of two control females were of spontaneous origin. Mild focal hemorrhage in the thymus of two control and one D3 animal is a terminal lesion without direct relation to the treatment.

Other microscopic findings were observed in either control animals only, or in both control and administered animals, or they were of a subsidiary nature, with no relation to the substances tested.

Tab. 18: Microscopic findings

Dose group: C D1 D2 D3
Sex: M F M F M F M F
Number of animals: 10 10 10 10 10 10 10 10
Organ findings:
Heart
- myocardial scar - 1 / / / / 2 -
Kidneys
- chronic pyelitis - - - - - - - 2
- corticomedullary
mineralization - - - - - - - 1
- hyaline casts 2 - 4 2 4 - 6 5
- hyaline droplets 5 - 10 - 10 - 10 -
- hydronephrosis 2 - 2 1 1 - 2 -
- medullary cyst 1 - - - - - - -
- tubular basophilia 1 - 10 - 10 - 10 2
- tubular necrosis - - 6 - 9 - 10 -
Larynx
- submucosal edema 8 3 / / / / 7 4
- chron. inflammation 6 1 / / / / 4 1
Liver
proc.papillaris
- hemorrhagic
infarction - - / / / / - 1
Lungs
- acute emphysema - 1 - - - - - -
- blood aspiration 3 1 2 4 3 3 5 6
- edema - - - - - - 1 -
- focal lipidosis 3 1 1 - 2 - 7 1
- focal chron. inflamm. - - - - - - 2 -
- focal hemorrhage 1 - - - - - 1 -
- osteoma racemosum - - - - 1 - - -
Lymph nodes
Cervical
- hemorrhage 2 1 / / / / - -
- hyperplasia 7 3 / / / / 5 5
Iliac
- hemosiderin - - / / / / 1 -
- hyperplasia - - / / / / 1 -
Prostate gland
- acute inflammation - NA / NA / NA 1 NA
Spinal cord
- hemorrhage 2 3 / / / / 2 -
Spleen
- venostasis - - / / / / 6 -
Thymus
- cyst - 2 / / / / - -
- focal hemorrhage 1 1 / / / / - 1
Thyroid gland
- squamous cell cyst - - / / / / - 1
Trachea
- blood aspiration 1 - / / / / 4 -
Urinary bladder
- chronic inflammation - - / / / / - 1
Uterus
- hydrometra NA 4 NA / NA / NA 3
- squamous cell cyst NA - NA / NA / NA 1

More detailed individual information are attached as pdf file.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Renal Immunohistochemistry: there was a statistically significant increase in tubular inclusions that stained immunohistochemically positively with antibodies against α2-microglobulin. The positive reaction was not dose-dependent but was significant at all doses.
Details on results:
The effects on the kidney in male rats are indicative for chronic progressive nephrosis, which is in line with the presence of hyaline droplets (identified to contain α2-microglobulin), the macroscopic effects and the proteinuria observed. This effect is treatment related but considered not relevant for humans.
The effects on the lungs are without a clear dose effect relationship.
Based on the effects on the lung, kidney, biochemical parameters in females the NOAEL for females can be set at 300 mg/kg bw. For male rats no NOAEL can be derived.

The increase of the testes weight (relative to body weight) as observed at 1000 mg/kg bw was not accompanied by macroscopic or histopathological findings. No findings on the female reproductive organs were found at any of the doses tested.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: non specific toxicity in females
Organ:
kidney
liver
lungs
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Based on the findings in this study the NOAEL is set at 300 mg/kg bw
Executive summary:

Male and female rats (10/sex/dose) were treated with the substance at 0, 100, 300 and 1000 mg/kg bw by gavage for 90 days according to OECD 408. No treatment findings on mortality, food intake and haematology were noted. At 1000 mg/kg bw liver and kidney weights were increased in males and females. In animals at 1000 mg/kg bw a time-related increased incidence of piloerection, decreased motility and laying in a rigid position was seen lasting up to 1.5 hours after dosing from day 28 onwards.

In females at 1000 mg/kg bw body weight (gain) was within control ranges , but slight effects on biochemistry (total protein (globulin), ALP, bile acids and cholesterol) were observed. Liver and kidney weights were increased.

Macroscopic and histopathological evaluation of these females showed enlarged and pale kidneys with hyaline casts. The lungs showed aspired blood and focal lipidosis. The NOAEL for females is therefore set at 300 mg/kg bw.

Males at 1000 mg/kg bw had a decreased body weight (gain). In addition these males showed decreased blood alkeline phosphatase levels and increased urinary ketone bodies and protein. At 300 and 1000 mg/kg kidney and liver weights increased in a dose dependent way. This was not accompanied by macroscopic and histological findings in the liver, but kidneys were found to be enlarged/pale containing hyaline droplets, hyaline casts, tubular basophilia and tubular necrosis indicative of chronic progressive nephrosis. Similar effects in the kidney were observed in all males at 100 and 300 mg/kg bw. These effects were confirmed to be related to α2-microglobulin (immunohistochemistry) and are therefore not relevant to humans. Effects in the lungs were similar to those found in females. Based on these findings no NOAEL could be set for males.

The NOAEL for females will be used as starting point for risk assessment.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-09-2012 to 30-11-2012
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar Han (IGS)
- Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 wks
- Weight at study initiation: Males: 296-335 g; Females: 197-223 g
- Housing:
three male animals in open macrolon cages type 2000P, TechniPlast (size slightly larger than GV-SOLAS Type IV)
females single housed with their litters in open macrolon cages type III.
- Diet : No. 1314 TPF (Altromin Spezialfutter GmbH & Co. KG, 32791 Lage), ad lib.
- Water: Sterilised community tap water, ad lib.
- Acclimation period: 5-9 days
- Pregnancy status females: Nulliparous, non-pregnant

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 03-09-2012 to 30-11-2012
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility in water was poor and good in corn oil
- Concentration in vehicle: 30.5 g/L, 91.2 g/L and 273.3 g/L
- Application volume: 4 mL/kg bw

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Stability of standard concentrations over a period of 7 days was investigated. All concentrations were within 96-102% of nominal.
Method GC/FID on ZB624 column. Two isomers were identified and the summation of both peaks was used for quantification
Duration of treatment / exposure:
Males: 14 days prior to mating; upto 14 days during mating and 14 days post-mating
Females: 14 days prior to mating; upto 14 days during mating; average 21 days of gestation; 4 days of lactation
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 111, 333, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a dose range finding study with increasing doses (upto 2000 mg/kg bw) over the 18 day study period. The animals showed toxic effects only at the highest dose level.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (IRWIN test) in a standard arena
Appearance (general status, physiology, autonomic functions, neurology, tonus, E1-E4, Motoric / exploration behaviour (M1-M2), Excitation (R) andAbnormal behaviour (A1-A4)

BODY WEIGHT: Yes
- Time schedule: weekly

FOOD CONSUMPTION: yes
Time schedule: weekly

WATER CONSUMPTION: Yes
Time schedule: weekly

HAEMATOLOGY: yes on 5 males and 5 females at end of premating period
Leukocytes, Erythrocytes, Haemoglobin , Haematocrit ,Mean corpuscular volume (MCV) , Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin conc. (MCHC), Thrombocytes, Reticulocytes, Neutrophil granulocytes, Lymphocytes, Monocytes, Eosinophils. Basophils

CLINICAL CHEMISTRY: yes on 5 males and 5 females at end of premating period
Alkaline phosphatase, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Cholesterol, Urea, Sodium, Potassium, Calcium, Chlorid, Glucose, Total protein, Albumin, Globulin, A/G ratio, Creatinine, Bile acids

OTHER:
Behavioural tests
Grip strength and beam walking: last exposure week on 5 males and 5 females
Sacrifice and pathology:
ORGAN WEIGHTS:
Thymus, Liver, Spleen, Kidney, Adrenals, Prostate, Epididymides, Testes, Ovaries

GROSS NECROPSY on all animals
Gross lesions, Oesophagus, Trachea, Thyroid, Stomach, Thymus, Liver, Spleen, Duodenum, Jejunum, Ileum (with Peyer’s patches), Cecum, Colon, Rectum, Lymph nodes(mesenteric), Kidney, Adrenals, Urinary bladder, sternum, spinal cord, whole brain, cerebrum, cerebellum, Peripheral nerve, Bone marrow, Pons, Skeletal muscle, Heart, Lungs, Vagina, Epididymides, Prostate/uterus/cervix, Testes/ovary, eye

HISTOPATHOLOGY:
ovaries, testes and epididymides of 5 animals of the high dose group and the control group
all organs of 5 males and 5 females of the high dose group and the control group
Statistics:
Anova with t-test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY (PARENTAL ANIMALS):
1000 mg/kg bw :1 female on day 14 , 1 male on day 19, 1 female on day 4 of lactation (gavage error, KIE)
none in other dose groups and controls

CLINICAL SIGNS/BEHAVIOURAL TESTS:
no treatment related effects

BODY WEIGHT (PARENTAL ANIMALS):
Males: sign. decrease during pre-mating and post-mating period at 1000 mg/kg bw;
Females: no treatment related effects, reduced during gestation in pregnant females of 1st mating (n=3)

FOOD CONSUMPTION (PARENTAL ANIMALS):
Males: increased at 1000 mg/kg bw
Females: no treatment related effects

WATER CONSUMPTION (PARENTAL ANIMALS):
Males: dose related increase at 111, 333 and 1000 mg/kg bw
Females: increased at 1000 mg/kg bw until day 14 of gestation

CLINICAL CHEMISTRY
Increased ALAT in males and females and decreased ALP in females at 1000 mg/kg bw
Slightly increased total protein in females of all dose groups

HAEMATOLOGY
Decreased Hb in females at 333 and 1000 mg/kg bw (no effects on MCH and MCHC)

ORGAN WEIGHTS (PARENTAL ANIMALS)
Kidney: increased in males at 333 and 1000 mg/kg bw
Liver: dose related increase in males (sign at 333 and 1000 mg/kg bw)
Thymus: decreased in males at 333 and 1000 mg/kg bw
Ovaries/uterus: increased at 1000 mg/kg bw

GROSS PATHOLOGY (PARENTAL ANIMALS)
no treatment related findings (all findings were incidental and within normal background)

HISTOPATHOLOGY (PARENTAL ANIMALS)
kidneys: hyaline droplets and/or casts in all males at 111, 333 and 1000 mg/kg bw
other findings were considered within normal ranges
Dose descriptor:
LOAEL
Effect level:
111 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: hyaline droplets and/or casts in the kidney
Key result
Dose descriptor:
NOAEL
Effect level:
333 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: effects at the high dose level were limited to decreased body weight (gain) during gestation, increased water consumption and a slight increase of the weight of the uteri
Critical effects observed:
not specified
Conclusions:
The NOAEL is 333 mg/kg bw based on toxicity and effects on reproduction
Executive summary:

Daily oral administration of the test substance to Wistar rats at dose levels of 111, 333, and 1000 mg/kg body weight over a period of 44 to 57 days resulted at the highest dose level in a reduction of the body weight gain and an increased water and food consumption in male animals. The water consumption was increased in females of the high dose group. Test item related changes in organ weight were found in the liver, the kidney and thymus of male animals. Furthermore, in male rats the histopathological examination indicated a nephrotoxic effect in all dose groups. At the highest dose level, the test item also showed a potential effect on the metabolism of the liver as well as an influence on uterus weight of female rats.

The NOAEL is 333 mg/kg bw for females. In males based on the effects on the kidney a LOAEL of 111 mg/kg bw was derived. As these effects are considered of less relevance for humans, the NOAEL used in risk assessment is set at 333 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the information available no classification according to CLP is considered necessary