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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.12. 2020 – 01.01. 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25th June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium salts of [hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid (4-7 K:1)
EC Number:
701-184-1
Molecular formula:
HMDTMP-4K C10H24K4N2O12P4 HMDTMP-5K C10H23K5N2O12P4 HMDTMP-6K C10H22K6N2O12P4 HMDTMP-7K C10H21K7N2O12P4
IUPAC Name:
Potassium salts of [hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid (4-7 K:1)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 - 7 weeks on arrival
- Weight at study initiation: males: 343-345 grams; females: 231-233 grams
- Fasting period before study: no
- Housing: 2 rats of the same sex in one plastic cage
- Diet (e.g. ad libitum): Free access to food ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 12-hour light/12-hour dark cycle
- Photoperiod (hrs dark / hrs light): 15 air changes per hour

IN-LIFE DATES: From: 29. 04. – 02. 05. 2020 To: 28. 07. – 31. 07. 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua pro iniectione
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulation was prepared by mixing with Aqua pro iniectione. The application forms of the test item, recalculated to 100 % w/w Total Active Acid HMDTMP-H were prepared daily just before administration.
Concentration level 34.6 mL/100 mL: 34.6 mL of the test item was measured by graduated cylinder (50 mL) to 150 mL volumetric beaker and replenish with 65.4 mL aqua pro iniectione by graduated cylinder (100 mL). After this the application form was stirred by magnetic stirrer for 30 min at 500 rpm by using magnetic stirring bar (3.5 cm).
Concentration level 0.35 mL/100 mL: Into 100 mL volumetric flask was measured 0.35 mL of the test item by syringe (0.5 mL) and replenished by the vehicle to required volume. After this the application form was stirred by magnetic stirrer for 30 min at 500 rpm by using magnetic stirring bar (3.5 cm).
The application forms of the test item, recalculated to 100 % w/w Total Active Acid HMDTMP-H were prepared daily just before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): aqua pro iniectione (water)
- Concentration in vehicle: 1 mL per 100 g of body weight

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the start of animal experiments the stability and homogeneity of application form was determined by means of measuring of a concentration K and P (mg/kg) in the test item application form at the test facility.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
active acid
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
active acid
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
active acid
No. of animals per sex per dose:
10 females and 10 males per group, 6 males and 6 females per satellite group
Details on study design:
- Dose selection rationale: The dose levels were based on a 28-day dose range-finding study, in which 0, 125, 500 and 1000 mg active acid/kg bw/day were administered daily by oral gavage to 6 male and 6 female rats for 28 days. In the dose-range finding study, there was no mortality or clinical signs of toxicity. The mean body weights and body weight increments for treated animals were comparable to the control group and were adequate for the species, sex and age of animals. Administration of the test item did not induce treatment-related toxicologically significant changes of the haematological parameters examined. No macroscopic changes were observed during necropsy of treated animals.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups: Satelite animals were included for the control and high dose group to check for reversibility of effects.
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): random
Positive control:
Not used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality; general clinical observations and health condition - daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before the first administration and then weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice per week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the 1st week of study and at the last week of administration and recovery period
- Dose groups that were examined: all animals at the start and only control and high dose group animals at the end of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 91st (main groups); 119th (satellite groups) day of study
- Anaesthetic used for blood collection: No
- Animals fasted: Yes (18 hours)
- How many animals: all animals
- Parameters checked in table [No.1] .

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study
- Animals fasted: Yes (18 hours)
- How many animals: all animals
- Parameters checked in table [No.2].

URINALYSIS: Yes
- Time schedule for collection of urine: 90th (main groups); 118th (satellite groups) day of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [No.3]

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the last week of administration and recovery period
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity, pupillary reflex, reactivity on auditory, visual, proprioceptive stimuli

IMMUNOLOGY: No

THYROID HORMONE ANALYSIS
- Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study
- Animals fasted: Yes (18 hours)
- How many animals: all animals
- Parameters checked: thyroxine (T4), triiodothyronine (T3) and rat Thyroid Stimulating Hormone (TSH)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4), on 91st (main group); 119th (satellite group) day of study. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland, thyroid gland and heart in control, all treated and satellite groups of animals were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

HISTOPATHOLOGY: Yes (see table 4). In the study the full histopathology of the preserved organs and tissues was performed for all high dose, control animals, recovery animals and animals with macroscopical changes.
Statistics:
The software Statgraphic® Centurion (version XVII, USA) was used. Males/females from the control group were compared with males/females from the three treated groups. Satellite males/females from the control group were compared with satellite males/females from the treated group. Results that were statistically significant were indicated using the probability values as follows: P < 0.05 (significant); P ≥ 0.05 (not significant). The parametric tests were used for statistical evaluation of the following parameters: body weight; selected haematology parameters; blood biochemistry; biometry of organs (absolute and relative weight). As the first step the test for normality (Shapiro-Wilk test) was performed. If data were not normally distributed the transformation of data was done (Box-Cox transformation). If data were normally distributed the variance check was performed (Levene´s test) to verify if standard deviations within each group were equal. One-Way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means. If significant differences were find, than the post hoc statistical testing was performed (Fisher's least significant difference - LSD test). If the presence of outliers was noted, the appropriate non-parametric test (Kruskal-Wallis Test, Mann-Whitney test) was chosen.
Non-parametric tests were used for statistical evaluation of following parameters: selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelets count). At first the Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group was used as global test, and then the two-groups Mann-Whitney test (probability level 0.05) was applied.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
In control animals and treated males and females of all dose levels and satellite groups no signs of disease were recorded during the check-in, acclimatisation and application period. During the observation period no changes of health status were noted in any of the animals. No clinical changes were recorded in any of the animals. No serious changes were found during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of all the animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male died at the 31st day of application at the dose level 125 mg active acid/kg bw/day due to intubation error (male No.23). There were no unscheduled deaths during all the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weights and necropsy body weights were similar in females of treated groups and control group for the whole application period. The body weights and necropsy body weights of satellite treated females were comparable with the control satellite females during the whole application period and recovery period. The body weights of males at 1000 mg active acid/kg bw/day were slightly, but not statistically significantly decreased compared to the control group for the whole administration period. Not statistically significantly decreased body weights and necropsy body weights were detected in satellite treated males compared to the control satellite group for the whole application and recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was comparable between treated and control groups in males and females. No effects were seen in food consumption in satellite groups.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was comparable between treated and control groups in males and females. No effects were seen in water consumption in satellite groups.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
During an examination performed before the start of the study and at the end of the study, no changes were noted.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: Parameters relating to red blood cell and white blood cell components and haemocoagulation parameters of treated males were comparable with control males. The fibrinogen concentration was statistically significantly decreased in all groups of treated males. The value of PT was statistically significantly decreased in 500 mg active acid/kg bw/day males. All haematological parameters were in range of historical control.
Satellite males: The relative count of reticulocytes was statistically significantly decreased in the 1000 mg active acid/kg bw/day group. The differential percentage count of monocytes was increased without statistical significance in satellite treated males in comparison with the control satellite group and was outside of the historical control range. This exceedance of the historical control range was due to a marked increase in the differential percentage count of monocytes in one male No.53 at this dose level. Other haematological parameters of satellite treated males were comparable to the satellite control males.
Females: Total leucocyte count was statistically significantly decreased in the 1000 mg active acid/kg bw/day group females. There were no statistically significant differences between the treated and control females for other white blood cell parameters. A statistically significant increase of RBC, together with an increased percentage HCT and haemoglobin concentration was noted in the 125 mg active acid/kg bw/day group. A statistically significantly increased platelet count was noted in the 1000 mg active acid/kg bw/day group. The relative reticulocyte count was statistically significantly decreased in the 125 and 1000 mg active acid/kg bw/day groups. Haemocoagulation parameter APTT was statistically significantly decreased in the 500 mg active acid/kg bw/day group. Fibrinogen concentration was significantly decreased in the 125 and 1000 mg active acid/kg bw/day groups. All haematological parameters were in range of historical control.
Satellite females: Statistically significant decreases in RBC, haemoglobin concentration and HCT were noted in satellite treated females compared with satellite control females. Other haematological parameters of satellite treated females were similar with the satellite control females. All haematological parameters were in range of historical control.
Overall, the differences were without dose response relationship and without correlation with other manifestation of toxicity.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: Statistically significantly increased urea concentration and decreased albumin concentration were recorded in the 1000 mg active acid/kg bw/day group. A statistically significantly decreased glucose concentration was recorded in the 500 mg active acid/kg bw/day group. A statistically significantly decreased potassium concentration was recorded in the 1000 mg active acid/kg bw/day group. Concentration of calcium was statistically significantly increased in the 125 mg active acid/kg bw/day group and statistically significantly decreased at 500 mg active acid/kg bw/day. Statistically significantly increased cholinesterase activity was recorded in the 1000 mg active acid/kg bw/day group. All changes are presented by comparison with the respective control group. Activities of ALT, AST and BUN exceeded the historical control range. However, for ALT and AST the marked increases were due to one male (No.44) and were without statistical significance. Others biochemical parameters were in range of historical control.
Satellite males: A statistically significantly increased concentration of inorganic phosphorus was recorded in treated satellite males compared with satellite control males. Other biochemical parameters of satellite treated males were similar with the satellite control males. All biochemical parameters were within the historical control range.
Females: A statistically significant increase in the activity of ALP was detected in the 1000 mg active acid/kg bw/day group. The value of T-Bil in treated females was decreased with statistical significance doses of 125 and 500 mg active acid/kg bw/day. Statistically significantly increased values of sodium ions were recorded in the 125 mg active acid/kg bw/day group, which were outside of the historical control range. All other biochemical parameters of treated females were similar with the control females and in the range of the historical control.
Satellite females: Statistically significantly increased glucose and chloride ion concentrations were recorded in high dose satellite females. Values of other parameters of the satellite high dose females were similar to satellite control females. All biochemical parameters were in range of historical control.
Overall, the differences were without dose response relationship and without correlation with other manifestation of toxicity.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: Statistically significant differences in pH and volume of urine were not detected. Presence of proteins, leukocytes and blood were recorded in treated males as well as in control males, which is why these findings were concluded as not associated with the application of the test item. The values of all other parameters of treated males were similar to control.
Satellite males: Statistically significant difference in pH of urine was not detected. Volume of urine was significantly decreased in treated males. The values of all other parameters of treated males were similar to the control.
Females: Statistically significantly increased pH was recorded for all doses. Volume of urine was statistically significantly increased in the 125 and 1000 mg active acid/kg bw/day groups. Presence of proteins and leukocytes in urine were also found only in these groups. Blood in urine was sporadically recorded in treated females as well as in control females, which is why these findings were concluded as not associated with the application of the test item. The values of all other parameters of treated females were similar to control.
Satellite females: Statistically significant differences in pH and volume of urine were not detected. The values of all parameters of treated satellite females were similar to the control satellite females.
All findings were not associated with the application of the test item.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Reactions to touch, noise, pain and pupillary reflex of treated males and females, and satellite animals were the same as in the control group. The number of upstanding, emiction and defecation of treated animals was without important changes. The time of grip of pectoral legs was slightly decreased in the 1000 mg active acid/kg bw/day males but without toxicological importance. The time of grip of pectoral legs was slightly increased in the 1000 mg/kg bw/day males but without toxicological importance. The time of grip of pectoral legs was slightly increased in treated females at 500 and 1000 mg active acid/kg bw/day but without toxicological importance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Males: The absolute testes weight in the 1000 mg active acid/kg bw/day group was statistically significantly increased compared to the control group. No other statistically significant differences in absolute weights of other organs of males were recorded. Relative testes weights were statistically significantly increased in the 1000 mg active acid/kg bw/day group. Statistically significantly increased liver relative weight was recorded in the 125 mg active acid/kg bw/day group. Relative weights of other organs of treated males were not statistically significantly different to those of control males.
Satellite males: The absolute thymus weight was statistically significantly decreased compared to the control group. No other statistically significant differences in absolute weights of other organs of males were recorded for satellite males. Statistically significantly decreased thymus relative weight in satellite treated males was recorded. The relative weight of other organs of the satellite treated and control males were similar.
Females: No statistically significant changes in absolute and relative organ weights of females were detected.
Satellite females: Absolute heart weight was statistically significantly decreased compared to the control group. No other statistically significant differences in absolute weights of other organs of females were recorded. Relative heart weight was statistically significantly decreased compared to the control group. Absolute weights of other organs of satellite females were not statistically significantly different to those of control females.
Overall, changes in weight of all organs usually associated with the effect of concern were not observed: no significant changes of red and white blood parameters and no histopathological findings.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Reduced testes and epididymis size were found in one control male and in two males of the lowest dose level. Male No. 23 died after application on 5th day of study at the dose level 125 mg active acid/kg bw/day(without pathological findings). This death is probably due to technical failure during application. There were no other findings in treated males and satellite group males at gross necropsy. In treated females and satellite females a non-pathological finding of uterus dilatation was detected in 2 of 0, 3 of 125, 1 of 500 and 3 of 1000 mg active acid/kg bw/day group treated females and 1 of control and 2 of 1000 mg active acid/kg bw/day satellite females. There were no macroscopical findings in treated and satellite females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The histopathological examination organs in treated and satellite group males and females showed only sporadical and in most cases spontaneous microscopical changes. These changes did not relate to the test item treatment. In treated and satellite females the uterus hydrometra, the non-inflammatory dilatation of the uterine wall, was recorded most often. Uterus hydrometra was recorded in 2 control, 3 females at 125, 1 female at 500 and 3 females at 1000 mg active acid/kg bw/day as well as in 1 satellite control and 2 satellite high dose females. Overall, no toxicologically significant findings were detected during the histopathological examination of organs in animals at the highest dose level and satellite treated animals. Sporadic findings in some organs were not related with test item treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Males: Statistically significant differences in concentrations of T4, T3 and TSH hormone were not detected. Means of the concentrations of hormones were similar for treated and control males.
Satellite males: Statistically significant differences in concentrations of T3 and TSH hormone were not detected. Means of the concentrations of T3 and TSH hormone were similar for treated and control.
Statistically significantly decreased concentrations of T4 hormone in satellite treated males was detected, but within the historical control range.
Females: Statistically significant differences in concentrations of T4 and T3 hormone were not detected. The mean concentrations of T4 and T3 hormone were similar in all treated females in comparison with the control females. Statistically significantly decreased concentration of TSH hormone at the dose levels 125 and 1000 mg/kg bw/day groups was detected, but within range of the historical controls.
Satellite females: Statistically significant differences in the concentrations of T3, T4 and TSH hormone were not detected. Mean concentrations of T3, T4 and TSH hormone were similar for high dose and control satellite females.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
active acid
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects observed.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

It follows from results of analyses (homogeneity and stability) that the solution of the test item in vehicle at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes from finalization of the application form preparation.

Table 1: Body Weight Males Group means and standard deviations (grams)

Time interval

0

125

500

1000

0 S

1000 S

Before application

343.98±11.35

345.98±18.12

345.89±14.78

344.38±13.22

345.50±11.05

344.57±7.21

1stweek

379.81±16.53

381.16±25.01

380.61±18.62

375.40±17.52

378.75±16.21

378.87±15.49

2ndweek    

407.46±18.92

406.71±24.81

406.35±22.52

398.82±21.43

408.45±19.44

409.57±20.74

3rdweek

431.50±17.78

432.41±34.72

428.38±24.83

422.43±27.70

432.43±25.47

435.68±23.51

4thweek

452.16±18.48

451.18±41.69

446.89±28.26

436.33±29.46

451.62±26.43

452.57±28.26

5thweek

466.45±18.23

453.23±30.64

461.08±30.04

453.85±32.12

465.47±27.06

469.22±33.75

6thweek

481.10±20.93

465.91±34.13

471.71±32.14

461.18±34.70

481.32±31.33

479.40±37.16

7thweek

489.98±19.17

477.61±34.47

481.28±32.53

471.25±38.66

490.50±33.88

490.98±37.69

8thweek

502.73±21.15

487.21±37.16

493.24±35.35

482.89±38.07

499.22±32.38

497.23±37.35

9thweek

512.08±19.04

498.29±39.05

502.89±33.93

489.89±38.12

506.15±29.64

504.65±40.82

10thweek

519.08±20.95

504.93±37.20

510.59±36.34

497.09±38.67

515.35±34.36

516.40±42.13

11thweek

526.88±21.86

512.02±38.92

518.32±37.54

504.41±36.63

522.77±33.87

523.50±43.51

12thweek

532.23±22.12

520.42±42.22

527.33±38.25

509.33±38.82

530.95±38.05

527.90±47.01

  13th week

542.84±20.53

527.76±42.64

531.80±36.67

514.83±38.00

539.33±37.34

533.48±50.97

14thweek

-

-

-

-

540.27±34.33

543.22±49.08

15thweek

-

-

-

-

539.50±36.06

549.70±50.13

16thweek

-

-

-

-

542.63±37.83

558.52±46.48

17th week

-

-

-

-

548.73±36.68

561.20±48.78

Total body weight increment*

163.03

146.6

151.19

139.43

169.98

182.33

Necropsy body weight

503.16±21.81

486.48±38.36

492.67±34.33

474.77±33.49

515.86±38.17

520.88±42.90

 Note:* 13thweek - 1stweek of study (satellite: 17thweek - 1stweek of study)

 

Table 2: Body weight increment males (group means (grams/animal/day)

Time interval

0

125

500

1000

0 S

1000 S

1stweek

5.12

5.03

4.96

4.43

4.75

4.90

2ndweek

3.95

3.65

3.68

3.35

4.24

4.39

3rdweek

3.43

3.67

3.15

3.37

3.43

3.73

4thweek

2.95

2.68

2.64

1.99

2.74

2.41

5thweek

2.04

0.29

2.03

2.50

1.98

2.38

6thweek

2.09

1.81

1.52

1.05

2.26

1.45

7thweek

1.27

1.67

1.37

1.44

1.31

1.65

8thweek

1.82

1.37

1.71

1.66

1.25

0.89

9thweek

1.34

1.58

1.38

1.00

0.99

1.06

10thweek

1.00

0.95

1.10

1.03

1.31

1.68

11thweek

1.11

1.01

1.10

1.05

1.06

1.01

12thweek

0.76

1.20

1.29

0.70

1.17

0.63

13thweek

1.77

1.22

0.75

0.92

1.40

0.93

14thweek

-

-

-

-

0.13

1.39

15thweek

-

-

-

-

-0.11

0.93

16thweek

-

-

-

-

0.45

1.26

17thweek

-

-

-

-

0.87

0.38

 

Table 3: Body Weight Females Group means and standard deviations (grams)

Time interval

0

125

500

1000

0 S

1000 S

Before application

232.38±12.13

232.67±19.18

232.01±8.02

233.30±6.48

231.40±9.32

233.35±11.37

1stweek

239.64±12.35

243.02±29.27

243.19±9.81

242.36±11.58

238.93±6.08

246.82±10.67

2ndweek

250.11±11.63

250.63±26.01

253.39±9.95

251.64±10.97

247.32±8.58

259.82±15.85

3rdweek

254.76±12.13

258.22±28.91

257.75±12.76

258.52±11.40

251.25±8.80

267.57±14.23

4thweek

265.06±12.90

267.88±27.06

271.60±12.36

267.97±11.22

266.18±9.36

274.48±17.15

5thweek

269.00±14.82

270.10±30.43

277.94±14.08

279.56±12.65

271.27±6.76

282.72±14.27

6thweek

274.64±11.40

275.14±26.58

282.61±14.08

281.97±11.65

271.28±6.40

283.20±14.49

7thweek

277.18±11.83

285.60±28.18

282.90±16.91

284.59±10.82

280.85±13.60

292.32±17.46

8thweek

286.80±20.00

289.66±32.24

291.13±16.27

290.63±10.07

282.97±11.65

298.07±18.94

9thweek

289.31±20.62

288.70±34.56

295.86±15.38

291.92±13.30

282.13±9.26

299.88±18.44

10thweek

292.74±13.71

295.62±33.03

300.25±15.84

298.96±8.05

287.55±11.23

302.05±16.65

11thweek

296.14±21.32

298.32±32.42

299.14±17.44

302.11±11.98

289.02±12.94

308.55±20.12

12thweek

297.49±18.75

305.41±33.15

307.94±16.63

307.19±10.17

299.18±10.78

314.43±23.49

13thweek

300.86±16.65

306.52±34.04

313.17±15.71

309.16±8.52

298.07±8.71

314.15±18.55

14thweek

-

-

-

-

300.45±11.09

318.18±22.20

15thweek

-

-

-

-

305.28±11.89

322.32±26.19

16thweek

-

-

-

-

327.55±25.27

306.25±13.34

17th week

-

-

-

-

303.87±9.12

325.63±22.52

Total body weight increment*

61.22

63.5

69.98

66.80

64.94

78.81

Necropsy body weight

274.09±12.95

281.82±31.28

282.14±13.95

282.07±9.56

284.39±10.22

299.67±21.42

Note:* 13thweek - 1stweek of study (satellite: 17thweek - 1stweek of study)

 

Table 4: Body Weight Increment Females Group mean (grams/animal/day)

Time interval

0

125

500

1000

0 S

1000 S

1stweek

1.04

1.48

1.60

1.29

1.08

1.92

2ndweek

1.50

1.09

1.46

1.33

1.20

1.86

3rdweek

0.66

1.08

0.62

0.98

0.56

1.11

4thweek

1.47

1.38

1.98

1.35

2.13

0.99

5thweek

0.56

0.32

0.91

1.66

0.73

1.18

6thweek

0.81

0.72

0.67

0.34

0.00

0.07

7thweek

0.36

1.49

0.04

0.37

1.37

1.30

8thweek

1.37

0.58

1.18

0.86

0.30

0.82

 9thweek

0.36

-0.14

0.68

0.18

-0.12

0.26

10thweek

0.49

0.99

0.63

1.01

0.77

0.31

11thweek

0.49

0.39

-0.16

0.45

0.21

0.93

12thweek

0.19

1.01

1.26

0.73

1.45

0.84

13thweek

0.56

0.19

0.87

0.33

-0.19

-0.05

14thweek

-

-

-

-

0.34

0.58

15thweek

-

-

-

-

0.69

0.59

16thweek

-

-

-

-

3.18

-2.30

17thweek

-

-

-

-

-3.38

2.77

 

Table 5: Food consumption males (grams/animal/day)

Time period

0

125

500

1000

0 S

1000 S

1sttime period

27.67

28.30

26.80

28.69

27.99

27.47

2ndtime period

26.04

26.76

26.65

25.73

25.94

26.07

3rdtime period

28.37

28.42

27.47

27.43

28.30

27.95

4thtime period

28.09

28.53

27.36

26.86

28.11

27.47

5thtime period

27.71

27.34

26.72

26.92

28.31

27.34

6thtime period

24.14

23.48

23.36

23.03

24.67

23.60

7thtime period

31.17

30.80

30.45

28.35

31.29

30.90

8thtime period

28.24

27.64

26.98

27.16

27.89

27.55

9thtime period

27.10

26.82

26.31

25.76

27.41

26.36

10thtime period

27.98

27.20

27.42

27.09

27.53

27.99

11thtime period

27.35

26.63

26.42

25.89

27.55

26.37

12thtime period

25.86

26.95

26.04

26.65

27.53

26.64

13thtime period

-

-

-

-

26.57

28.48

14thtime period

-

-

-

-

23.69

23.34

15thtime period

-

-

-

-

29.58

31.04

16thtime period

-

-

-

-

27.46

28.22

Note:Time period: 1 time period = 7 days (week)by reason of successive start and end of application of groups

 

Table 6: Food consumption females (grams/animal/day)

Time period

0

125

500

1000

0 S

1000 S

1sttime period

21.43

20.59

20.86

22.07

22.90

21.71

2ndtime period

17.98

19.58

19.63

19.09

18.19

18.69

3rdtime period

12.26

15.06

16.47

12.38

14.53

13.83

4thtime period

25.27

26.80

26.15

27.31

26.65

26.24

5thtime period

19.02

18.64

19.05

16.97

17.65

19.58

6thtime period

15.01

16.80

15.86

17.61

16.28

17.06

7thtime period

22.06

23.76

23.55

24.19

22.05

23.41

8thtime period

19.98

22.30

20.09

24.63

21.97

21.54

9thtime period

18.77

19.20

19.00

16.56

16.20

19.46

10thtime period

18.64

16.39

21.36

18.75

21.05

20.41

11thtime period

18.82

20.55

19.33

20.34

19.06

19.98

12thtime period

17.97

20.13

20.10

19.10

17.67

20.73

13thtime period

-

-

-

-

19.11

20.62

14thtime period

-

-

-

-

15.88

17.99

15thtime period

-

-

-

-

22.30

23.34

16thtime period

-

-

-

-

19.45

21.45

Note:Time period: 1 time period = 7 days (week)by reason of successive start and end of application of groups

 

Table 7: Food conversion ratio (%) males

Group code

0

125

500

1000

0 S

1000 S

1sttime period

14.28

12.90

13.73

11.68

15.15

15.98

2ndtime period

13.17

13.71

11.82

13.10

13.22

14.31

3rdtime period

10.40

9.43

9.61

7.25

9.68

8.62

4thtime period

7.26

1.02

7.42

9.31

7.04

8.66

5thtime period

7.54

6.62

5.69

3.90

7.98

5.30

6thtime period

5.26

7.11

5.86

6.25

5.31

6.99

7thtime period

5.84

4.45

5.62

5.86

3.99

2.88

8thtime period

4.75

5.72

5.11

3.68

3.55

3.85

9thtime period

3.69

3.54

4.18

4.00

4.78

6.37

10thtime period

3.95

3.71

4.01

3.88

3.85

3.61

11thtime period

2.78

4.51

4.88

2.70

4.25

2.39

12thtime period

6.47

4.53

2.88

3.45

5.09

3.49

13thtime period

-

-

-

-

0.49

4.88

14thtime period

-

-

-

-

-0.46

3.98

15thtime period

-

-

-

-

1.52

4.06

16thtime period

-

-

-

-

3.17

1.35

Note:Time period: 1 time period = 7 days (week)by reason of successive start and end of application of groups

 

Table 8: Food conversion ratio (%) females

Group code

0

125

500

1000

0 S

1000 S

1sttime period

7.00

5.29

7.00

6.03

5.24

8.57

2ndtime period

3.67

5.52

3.16

5.13

3.08

5.94

 3rdtime period

11.99

9.16

12.02

10.90

14.66

7.16

4thtime period

2.22

1.19

3.48

6.08

2.74

4.50

5thtime period

4.26

3.86

3.52

2.00

0.01

0.36

6thtime period

2.40

8.87

0.25

2.10

8.42

7.62

7thtime period

6.21

2.44

5.01

3.56

1.36

3.50

8thtime period

1.80

-0.63

3.38

0.73

-0.55

1.21

9thtime period

2.61

5.16

3.32

6.10

4.75

1.59

10thtime period

2.63

2.38

-0.75

2.40

1.00

4.56

11thtime period

1.01

4.91

6.52

3.59

7.61

4.20

12thtime period

3.12

0.94

4.33

1.73

-1.08

-0.24

13thtime period

-

-

-

-

1.78

2.81

14thtime period

-

-

-

-

4.35

3.28

15thtime period

-

-

-

-

14.26

-9.85

16thtime period

-

-

-

-

-17.38

12.91

Note:Time period: 1 time period = 7 days (week)by reason of successive start and end of application of groups

 

 

Table 9: Water consumption males group mean (mL/animal/day)

Group code

0

125

500

1000

0 S

1000 S

1sttime period

61.71

64.00

54.14

60.43

58.81

61.90

2ndtime period

62.43

61.14

51.29

60.43

63.57

61.67

3rdtime period

60.29

58.14

50.43

58.86

63.10

60.95

4thtime period

58.86

59.57

50.71

60.71

67.62

60.71

5thtime period

62.43

61.27

52.29

59.00

65.48

66.67

6thtime period

60.86

59.68

50.00

58.43

67.62

59.52

7thtime period

65.71

67.94

55.57

60.29

64.05

60.00

8thtime period

60.29

59.68

54.14

59.71

65.71

62.38

9thtime period

63.86

61.90

49.57

56.71

67.38

62.14

10thtime period

63.29

60.63

48.71

58.43

64.05

62.86

11thtime period

61.29

59.05

48.71

54.86

66.43

60.24

12thtime period

66.86

62.06

51.71

62.86

70.00

63.33

13thtime period

-

-

-

-

61.19

54.52

14thtime period

-

-

-

-

64.29

58.10

15thtime period

-

-

-

-

65.95

59.52

16thtime period

-

-

-

-

62.62

60.71

Note:Time period: 1 time period = 7 days (week)by reason of successive start and end of application of groups

 

Table 10: Water consumption females group mean (mL/animal/day)

Group code

0

125

500

1000

0 S

1000 S

1sttime period

47.73

46.71

46.57

46.00

46.43

48.10

2ndtime period

45.23

43.57

43.43

44.00

44.05

47.14

3rdtime period

42.43

41.71

43.86

47.43

44.05

42.86

4thtime period

41.86

42.71

44.14

45.00

45.57

45.48

5thtime period

44.00

43.86

43.00

46.00

44.29

47.14

6thtime period

40.29

45.71

42.86

44.14

42.62

44.29

7thtime period

42.57

43.00

41.71

42.29

39.29

40.00

8thtime period

45.86

45.00

42.29

40.43

39.05

47.62

9thtime period

41.14

43.29

43.00

44.43

41.90

43.81

10thtime period

40.71

45.86

43.71

44.43

42.86

44.71

11thtime period

37.71

45.00

43.00

41.14

41.67

45.24

12thtime period

40.14

48.29

46.57

48.86

44.05

47.62

13thtime period

-

-

-

-

37.86

43.81

14thtime period

-

-

-

-

40.00

44.29

15thtime period

-

-

-

-

41.67

44.05

16thtime period

-

-

-

-

43.33

45.24

Note:Time period: 1 time period = 7 days (week)by reason of successive start and end of application of groups

 

Table 11: Health Condition Males

Time interval

0

125

500

1000

0 S

1000 S

Acclimatization

1/10

1/10

1/10

1/10

1/6

1/6

1stweek

1/10

1/10

1/10

1/10

1/6

1/6

2ndweek

1/10

1/10

1/10

1/10

1/6

1/6

3rdweek

1/10

1/10

1/10

1/10

1/6

1/6

4thweek

1/10

1/10

1/10

1/10

1/6

1/6

5thweek

1/10

1/9*

1/10

1/10

1/6

1/6

6thweek

1/10

1/9

1/10

1/10

1/6

1/6

7thweek

1/10

1/9

1/10

1/10

1/6

1/6

8thweek

1/10

1/9

1/10

1/10

1/6

1/6

9thweek

1/10

1/9

1/10

1/10

1/6

1/6

10thweek

1/10

1/9

1/10

1/10

1/6

1/6

11thweek

1/10

1/9

1/10

1/10

1/6

1/6

12thweek

1/10

1/9

1/10

1/10

1/6

1/6

13thweek

1/10

1/9

1/10

1/10

1/6

1/6

14thweek

-

-

-

-

1/6

1/6

15thweek

-

-

-

-

1/6

1/6

16thweek

-

-

-

-

1/6

1/6

17thweek

-

-

-

-

1/6

1/6

Note:  1 – normal physiological appearance

           1/10 finding/ number of animals with finding

           *male No. 23 – died during the study at the 5thweek of application

 

Table 12: Health Condition Females

Time interval

0

125

500

1000

0 S

1000 S

Acclimatization

1/10

1/10

1/10

1/10

1/6

1/6

1stweek

1/10

1/10

1/10

1/10

1/6

1/6

2ndweek

1/10

1/10

1/10

1/10

1/6

1/6

3rdweek

1/10

1/10

1/10

1/10

1/6

1/6

4thweek

1/10

1/10

1/10

1/10

1/6

1/6

5thweek

1/10

1/10

1/10

1/10

1/6

1/6

6thweek

1/10

1/10

1/10

1/10

1/6

1/6

7thweek

1/10

1/10

1/10

1/10

1/6

1/6

8thweek

1/10

1/10

1/10

1/10

1/6

1/6

9thweek

1/10

1/10

1/10

1/10

1/6

1/6

10thweek

1/10

1/10

1/10

1/10

1/6

1/6

11thweek

1/10

1/10

1/10

1/10

1/6

1/6

12thweek

1/10

1/10

1/10

1/10

1/6

1/6

13thweek

1/10

1/10

1/10

1/10

1/6

1/6

14thweek

-

-

-

-

1/6

1/6

15thweek

-

-

-

-

1/6

1/6

16thweek

-

-

-

-

1/6

1/6

17thweek

-

-

-

-

1/6

1/6

Note:  1 – normal physiological appearance

           1/10 finding/ number of animals with finding

Table 13: Clinical Observation Males

Time interval

0

125

500

1000

0 S

1000 S

1stweek

1/10

1/10

1/10

1/10

1/6

1/6

2ndweek

1/10

1/10

1/10

1/10

1/6

1/6

3rdweek

1/10

1/10

1/10

1/10

1/6

1/6

4thweek

1/10

1/10

1/10

1/10

1/6

1/6

5thweek

1/10

1/9*

1/10

1/10

1/6

1/6

6thweek

1/10

1/9

1/10

1/10

1/6

1/6

7thweek

1/10

1/9

1/10

1/10

1/6

1/6

8thweek

1/10

1/9

1/10

1/10

1/6

1/6

9thweek

1/10

1/9

1/10

1/10

1/6

1/6

10thweek

1/10

1/9

1/10

1/10

1/6

1/6

11thweek

1/10

1/9

1/10

1/10

1/6

1/6

12thweek

1/10

1/9

1/10

1/10

1/6

1/6

13thweek

1/10

1/9

1/10

1/10

1/6

1/6

 

Note:  1 – nomal physiological appearance

           1/10 finding/ number of animals with finding

           *male No. 23 – died during the study at the 5thweek of application

 

 Table 14: Clinical Observation Females

Time interval

0

125

500

1000

0 S

1000 S

1stweek

1/10

1/10

1/10

1/10

1/6

1/6

2ndweek

1/10

1/10

1/10

1/10

1/6

1/6

3rdweek

1/10

1/10

1/10

1/10

1/6

1/6

4thweek

1/10

1/10

1/10

1/10

1/6

1/6

5thweek

1/10

1/10

1/10

1/10

1/6

1/6

6thweek

1/10

1/10

1/10

1/10

1/6

1/6

7thweek

1/10

1/10

1/10

1/10

1/6

1/6

8thweek

1/10

1/10

1/10

1/10

1/6

1/6

9thweek

1/10

1/10

1/10

1/10

1/6

1/6

10thweek

1/10

1/10

1/10

1/10

1/6

1/6

11thweek

1/10

1/10

1/10

1/10

1/6

1/6

12thweek

1/10

1/10

1/10

1/10

1/6

1/6

13thweek

1/10

1/10

1/10

1/10

1/6

1/6

 

Note:  1 – normal physiological appearance         

           1/10 finding/ number of animals with finding

 

Table 15: Detailed Clinical Observation Males

Observation

0

125

500

1000

0 S

1000 S

Skin

1

1

1

1

1

1

Hair - piloerection

1

1

1

1

1

1

Eyes (pupils)

1

1

1

1

1

1

Lacrimation

1

1

1

1

1

1

Visible mucose membranes

1

1

1

1

1

1

Secretions

1

1

1

1

1

1

Excrements

1

1

1

1

1

1

Respiration

1

1

1

1

1

1

Poise

1

1

1

1

1

1

Gait

1

1

1

1

1

1

Reaction to handling

1

1

1

1

1

1

Clonic-tonic movements

1

1

1

1

1

1

Stereotypes

0

0

0

0

0

0

Vocalisation

0

0

0

0

0

0

Bizarre behaviour

0

0

0

0

0

0

Salivation 

1

1

1

1

1

1

Other findings

0

0

0

0

0

0

 

Note:  0 - not found,

          1 - natural reaction (physiological, without abnormalities)

 

Table 16: Detailed Clinical Observation Females

Observation

0

125

500

1000

0 S

1000 S

Skin

1

1

1

1

1

1

Hair – piloerection

1

1

1

1

1

1

Eyes (pupils)

1

1

1

1

1

1

Lacrimation

1

1

1

1

1

1

Visible mucose membranes

1

1

1

1

1

1

Secretions

1

1

1

1

1

1

Excrements

1

1

1

1

1

1

Respiration

1

1

1

1

1

1

Poise

1

1

1

1

1

1

Gait

1

1

1

1

1

1

Reaction to handling

1

1

1

1

1

1

Clonic-tonic movements

1

1

1

1

1

1

Stereotypes

0

0

0

0

0

0

Vocalisation

0

0

0

0

0

0

Bizarre behaviour

0

0

0

0

0

0

Salivation

1

1

1

1

1

1

Other findings

0

0

0

0

0

0

 

Note: 0-without reaction

        1 – natural reaction

Table 17: Functional observation Males

Observation

Unit

Group code

Group code

Group code

Group code

Group code

Group code

0

125

500

1000

0 S

1000 S

Sensory activity

 

 

 

 

 

 

 

1. Reaction to approximation

1-4

1

1

1

1

1

1

2. Reaction to contact point

1-4

1

1

1

1

1

1

3. Reaction to noise

1-4

1

1

1

1

1

1

4. Pupillary reflex

1-4

1

1

1

1

1

1

5. Reaction to pain

1-4

1

1

1

1

1

1

Motor activity

 

 

 

 

 

 

 

6. Upstanding

3 min

21.30

20.22

20.40

20.90

20.33

19.50

7. Emiction

3 min

0.80

0.67

0.50

0.60

0.33

0.50

8. Defecation

3 min

0.80

0.67

0.80

0.40

0.17

0.67

Grip strength

 

 

 

 

 

 

 

9. Pectoral legs – time of grip

Second

2.23

2.14

2.19

1.72

1.81

2.67

10. Stretch the pelvis legs

yes/no

yes

yes

yes

yes

yes

yes

11. Pectoral legs

Newton

1.66

2.49

1.32

2.14

1.90

2.30

12. Pelvis legs

Newton

0.44

0.42

0.30

0.34

0.28

0.40

 

Note:  Line 1-5: grading scale:1= natural reaction;2= mild;3= increased;4= without reaction

           Line 6-8: number of upstanding, emiction and defecation within 3 minutes

           Line 10: stretch the hind legs on fall from a height - a natural reaction (yes / no)

          Line 11-12: grip power in Newton

 

Table 18: Functional observation Females

Observation

Unit

Group code

Group code

Group code

Group code

Group code

Group code

0

125

500

1000

0 S

1000 S

Sensory activity

 

 

 

 

 

 

 

1. Reaction to approximation

1-4

1

1

1

1

1

1

2. Reaction to contact point

1-4

1

1

1

1

1

1

3. Reaction to noise

1-4

1

1

1

1

1

1

4. Pupillary reflex

1-4

1

1

1

1

1

1

5. Reaction to pain

1-4

1

1

1

1

1

1

Motor activity

 

 

 

 

 

 

 

6. Upstanding

3 min

20.60

20.10

21.20

20.90

20.67

21.83

7. Emiction

3 min

0.40

1.00

0.30

0.30

0.83

1.50

8. Defecation

3 min

0.40

0.80

0.90

0.70

1.00

0.67

Grip strength

 

 

 

 

 

 

 

9. Pectoral legs – time of grip

Second

1.98

1.54

2.62

2.59

2.04

2.11

10. Stretch the pelvis legs

yes/no

yes

yes

yes

yes

yes

yes

11. Pectoral legs

Newton

1.99

1.67

1.35

1.28

1.55

1.90

12. Pelvis legs

Newton

0.48

0.25

0.35

0.28

0.20

0.17

 

Note:  Line 1-5: grading scale:1= natural reaction;2= mild;3= increased;4= without reaction

           Line 6-8: number of upstanding, emiction and defecation within 3 minutes

           Line 10: stretch the hind legs on fall from a height - a natural reaction (yes / no)

Table 19: Haematological examination males

Parameters

Unit

0

125

500

1000

0 S

1000 S

Values

WBC -Total leucocyte count

109/L

8.64

 8.75

±1.34

9.32

8.96

±0.81

10.16

9.89

±1.08

8.53

8.95

±2.15

8.74

9.08

±1.56

7.29

7.53

±0.88

  Median

Mean

+SD

RBC -Total erythrocyte count

1012/L

8.13

8.06

±0.77

8.21

8.24

±0.50

7.91

7.85

±0.51

8.02

7.98

±0.32

8.26

8.35

±0.51

8.49

8.32

±0.41

  Median

Mean

+SD

Haemoglobin

g/100mL

15.15

14.92

±0.88

15.20

15.19

±0.61

14.40

14.46

±0.75

14.55

14.61

±0.33

14.45

14.55

±0.39

14.60

14.55

±0.64

Median

Mean

+SD

HCT

Haematocrite

%

40.25

40.08

±3.47

41.60

41.39

±1.64

39.20

39.51

±2.63

40.30

40.26

±0.98

41.45

41.52

±1.39

41.30

41.78

±1.67

Median

Mean

+SD

MCV -Mean corpuscular volume

Fl

49.25

49.85

±1.58

49.80

50.37

±2.27

51.00

50.44

±1.28

50.35

50.56

±1.84

50.15

49.83

±1.48

50.55

50.32

±1.34

  Median

Mean

+SD

Platelet count

 109/L

935.5

929.8

±104.47

947.0

948.1

±103.47

928.5

933.9

±105.47

853.5

878.4

±135.93

922.00

878.50

±184.42

1075.00

1034.33

±118.89

Median

Mean

+SD

 

Lymphocytes

 

% WBC

69.89

68.83

±4.01

68.83

66.63

±7.27

65.05

64.16

±11.62

64.51

65.91

±5.59

72.33

69.39

±9.37

66.44

62.61

±15.01

Median

Mean

+SD

 

Monocytes

 

% WBC

7.56

6.57

±1.95

6.22

8.85

±8.70

6.08

9.03

±8.35

5.63

6.12

±1.36

6.43

6.46

±0.45

7.29

11.38*

±11.45

Median

Mean

+SD

Neutrophiles

% WBC

22.20

22.08

±3.45

22.08

22.50

±5.71

25.88

24.56

±5.87

26.64

25.61

±4.94

19.52

21.72

±8.71

23.91

23.23

±5.89

Median

Mean

+SD

Eosinophiles

% WBC

2.21

2.37

±0.72

1.79

1.88

±0.66

2.05

2.16

±0.56

2.29

2.20

±0.57

2.40

2.25

±0.97

2.74

2.69

±0.62

Median

Mean

+SD

Basophiles

% WBC

0.11

0.15

±0.11

0.16

0.14

±0.10

0.06

0.09

±0.09

0.17

0.17

±0.06

0.16

0.18

±0.17

0.09

0.10

±0.06

Median

Mean

+SD

APTTActivated Partial Thrombo-

plastin Time

s

17.35

19.08

±5.32

17.80

19.24

±4.09

17.20

18.64

±5.79

16.75

17.11

±2.09

18.50

18.33

±2.81

17.95

18.07

±1.97

Median

Mean

+SD

Fibrinogen

g/L

1.89

1.90

±0.10

1.74

1.74

±0.12

1.80

1.75

±0.22

1.75

1.74

±0.10

2.11

2.07

±0.12

1.95

1.97

±0.10

Median

Mean

+SD

PT

Prothrombin Time

s

23.40

22.82

±2.11

23.60

23.91

±1.69

20.10

20.13

±1.99

23.05

23.06

±1.49

21.95

22.30

±1.89

24.05

23.73

±1.52

Median

Mean

+SD

Reticulocytes

%

2.35

2.45

±0.63

2.50

2.56

±0.58

2.50

2.50

±0.31

2.05

2.20

±0.45

3.60

3.67

±0.26

3.10

3.17

±0.32

Median

Mean

+SD

Note:  male No. 23 – died during study        

           *value out of the historical control range

 

Table 20: Haematological examination females

Parameters

Units

0

125

500

1000

0 S

1000 S

Values

WBC -Total leucocyte count

109/L

4.93

5.29

±1.29

4.19

4.26

±0.74

4.23

4.26

±0.92

4.12

4.08

±0.86

4.14

4.52

±1.20

4.23

4.58

±1.31

Median

Mean

+SD

RBC -Total erythrocyte count

1012/L

7.16

7.11

±0.34

7.48

7.57

±0.43

7.20

7.35

±0.44

7.31

7.16

±0.37

7.57

7.56

±0.25

7.09

7.13

±0.34

Median

Mean

+SD

Haemoglobin

g/100mL

14.40

14.34

±0.33

15.05

15.15

±0.56

14.50

14.39

±0.52

14.25

14.18

±0.45

14.50

14.43

±0.27

13.85

13.80

±0.23

Median

Mean

+SD

HCT

Haematocrite

%

38.35

37.95

±1.42

40.30

40.35

±1.63

39.50

39.03

±2.11

38.65

37.98

±1.52

40.05

40.28

±0.79

38.60

38.35

±0.84

Median

Mean

+SD

MCV -Mean corpuscular volume

Fl

53.30

53.50

±1.09

53.65

53.42

±1.35

53.25

53.15

±1.37

52.95

53.15

±1.83

53.00

53.38

±1.23

53.95

53.90

±1.80

Median

Mean

+SD

Platelet count

 109/L

1065.5

1025.5

±158.28

935.5

937.7

±99.02

985.0

946.4

±120.03

844.0

862.7

±81.85

856.00

883.33

±138.05

765.00

782.33

±123.18

Median

Mean

+SD

 

Lymphocytes

 

% WBC

79.57

77.40

±5.79

75.08

72.68

±6.89

77.41

75.31

±6.60

80.33

77.03

±7.96

77.31

78.75

±2.60

77.67

75.74

±5.38

Median

Mean

+SD

 

Monocytes

 

% WBC

7.47

10.27

±5.55

7.34

12.15

±8.41

9.23

11.46

±6.94

4.23

6.23

±5.41

7.81

7.93

±2.05

6.83

8.72

±5.70

Median

Mean

+SD

Neutrophiles

% WBC

12.18

10.99

±8.10

14.55

13.57

±8.45

12.75

12.12

±9.16

13.01

14.93

±8.15

10.62

11.18

±1.92

13.90

13.16

±6.23

Median

Mean

+SD

Eosinophiles

% WBC

1.61

1.28

±1.12

1.67

1.53

±1.26

1.20

1.04

±0.98

1.27

1.69

±1.08

2.00

1.99

±0.73

2.55

2.30

±1.38

Median

Mean

+SD

Basophiles

% WBC

0.06

0.06

±0.05

0.07

0.07

±0.06

0.05

0.07

±0.09

0.13

0.13

±0.07

0.11

0.15

±0.15

0.08

0.08

±0.04

Median

Mean

+SD

APTTActivated Partial Thrombo-

plastin Time

s

16.90

17.13

±0.65

17.70

18.02

±1.42

16.00

15.85

±1.56

16.65

16.15

±1.63

17.00

17.40

±1.37

16.70

17.03

±0.90

Median

Mean

+SD

Fibrinogen

g/L

1.39

1.43

±0.12

1.34

1.31

±0.13

1.44

1.43

±0.10

1.34

1.29

±0.11

1.59

1.59

±0.11

1.62

1.58

±0.09

Median

Mean

+SD

PT

Prothrombin Time

s

24.65

24.29

±2.08

25.35

25.39

±1.39

23.80

23.55

±2.02

24.35

24.15

±1.67

26.00

25.70

±1.39

25.50

25.27

±1.57

Median

Mean

+SD

Reticulocytes

%

2.50

2.55

±0.46

1.95

2.01

±0.36

2.50

2.53

±0.50

2.30

2.07

±0.32

2.90

2.83

±0.36

2.85

2.87

±0.39

Median

Mean

+SD

 

Note:grey field= values statistically significant (p<0.05)

 

Table 21: Biochemical examination males

Parameters

Units

0

125

500

1000

0 S

1000 S

Values

T-Pro

g/L

66.51

67.44

±3.22

69.61

69.54

±2.46

67.41

67.40

±1.85

66.75

66.58

±2.03

66.91

65.87

±3.55

66.73

66.73

±3.07

  Median

Mean

+SD

ALP

mkat/L

1.28

1.39

±0.32

1.35

1.36

±0.31

1.47

1.52

±0.45

1.42

1.52

±0.36

1.41

1.43

±0.25

1.51

1.50

±0.24

  Median

Mean

+SD

T-Chol

mmol/L

1.85

1.98

±0.45

1.90

2.06

±0.36

2.00

1.97

±0.30

1.90

2.02

±0.34

2.10

2.08

±0.24

1.95

2.12

±0.48

Median

Mean

+SD

TG

  mmol/L

0.87

0.89

±0.27

0.78

0.91

±0.49

0.77

0.84

±0.32

1.03

1.05

±0.41

1.01

1.04

±0.34

0.68

0.79

±0.34

Median

Mean

+SD

ALT

mkat/L

0.83

0.82

±0.19

0.74

0.99

±0.58

0.78

0.78

±0.12

0.77

1.22*

±1.34

0.80

1.04

±0.72

0.80

0.87

±0.18

  Median

Mean

+SD

AST

mkat/L

2.07

2.12

±0.42

1.86

2.21

±0.81

2.67

2.74

±0.56

2.18

3.30*

±3.02

1.66

2.03

±1.07

1.62

1.81

±0.37

Median

Mean

+SD

Crea

mmol/L

25.55

26.23

±2.21

27.50

27.58

±2.88

28.55

28.42

±2.56

27.90

28.66

±2.73

28.15

27.92

±3.09

25.10

25.33

±2.37

Median

Mean

+SD

BUN

mmol/L

6.93

6.95

±1.03

7.52

7.43

±0.53

7.13

7.13

±0.66

8.48

9.08*

±1.90

6.42

6.39

±0.15

6.47

6.62

±1.10

Median

Mean

+SD

ALB

g/L

43.33

43.62

±1.47

44.06

44.32

±1.07

42.96

42.92

±1.01

42.50

42.46

±1.04

41.21

41.27

±1.54

42.30

42.16

±1.48

Median

Mean

+SD

T-BIL

mmol/L

2.52

2.41

±0.38

2.56

2.47

±0.42

2.11

2.15

±0.33

2.49

2.36

±0.41

2.48

2.44

±0.50

1.95

2.10

±0.35

Median

Mean

+SD

GLU

mmol/L

5.67

5.54

±0.61

5.56

5.73

±0.61

4.64

4.87

±0.56

5.82

5.67

±0.83

5.88

5.93

±0.53

5.32

5.71

±0.97

Median

Mean

+SD

Ca

mmol/L

2.57

2.58

±0.09

2.64

2.66

±0.08

2.47

2.47

±0.07

2.52

2.54

±0.05

2.73

2.72

±0.09

2.69

2.71

±0.06

Median

Mean

+SD

IP

mmol/L

2.22

2.22

±0.14

2.17

2.14

±0.10

2.13

2.14

±0.09

2.20

2.22

±0.14

1.81

1.82

±0.11

1.99

2.00

±0.06

Median

Mean

+SD

CHE

mkat/L

6.45

6.84

±1.00

7.20

7.45

±1.27

7.42

6.90

±1.86

9.33

9.21

±1.53

5.81

6.01

±1.77

5.78

5.94

±1.88

Median

Mean

+SD

BA

mmol/L

53.78

51.74

±25.08

61.35

63.12

±27.65

45.50

44.91

±23.02

45.70

56.48

±30.26

46.43

45.44

±14.21

62.92

66.90

±41.27

Median

Mean

+SD

LDL Direct

mmol/L

0.50

0.54

±0.15

0.53

0.55

±0.10

0.56

0.55

±0.10

0.53

0.54

±0.10

0.52

0.51

±0.09

0.48

0.53

±0.12

Median

Mean

+SD

HDL Direct

mmol/L

0.50

0.51

±0.05

0.52

0.52

±0.05

0.50

0.49

±0.05

0.53

0.53

±0.04

0.46

0.46

±0.03

0.48

0.47

±0.07

Median

Mean

+SD

 

Na

 

mmol/L

141.50

141.50

±1.84

141.00

141.67

±1.58

141.00

141.30

±1.25

141.00

140.90

±1.85

139.00

139.67

±1.21

140.00

139.67

±1.86

Median

Mean

+SD

 

K

 

mmol/L

3.90

3.99

±0.22

3.90

3.91

±0.15

3.90

3.87

±0.18

3.60

3.65

±0.18

3.90

3.92

±0.17

3.95

3.90

±0.21

Median

Mean

+SD

 

Cl

 

mmol/L

102.00

101.70

±0.67

103.00

102.78

±1.79

102.50

102.30

±1.34

103.00

102.70

±1.77

102.50

102.17

±0.98

103.00

102.83

±0.98

Median

Mean

+SD

 

Note:grey field= values statistically significant (P < 0.05)

         *value out of the historical control range

         male No. 23 – died during study

Table 22: Biochemical examination females

Parameters

Units

0

125

500

1000

0S

1000S

Values

T-Pro

g/L

69.99

70.02

±5.12

71.16

70.63

±4.28

67.05

67.63

±2.18

67.51

67.33

±5.77

68.73

68.39

±3.27

67.92

67.86

±2.91

  Median

Mean

+SD

ALP

mkat/L

0.60

0.66

±0.17

0.69

0.69

±0.13

0.57

0.59

±0.13

0.91

0.88

±0.17

0.67

0.72

±0.20

0.54

0.58

±0.12

  Median

Mean

+SD

T-Chol

mmol/L

2.05

2.31

±0.63

1.90

1.89

±0.28

2.05

2.09

±0.52

2.10

2.07

±0.55

2.45

2.45

±0.15

2.45

2.42

±0.15

Median

Mean

+SD

TG

  mmol/L

1.00

0.98

±0.47

0.81

0.78

±0.40

1.04

1.13

±0.30

1.21

1.16

±0.45

0.97

0.97

±0.36

1.34

1.42

±0.51

Median

Mean

+SD

ALT

mkat/L

0.64

0.71

±0.21

0.83

1.01

±0.52

0.63

1.07

±1.35

0.65

0.68

±0.14

0.77

0.77

±0.09

0.77

0.98

±0.65

  Median

Mean

+SD

AST

mkat/L

1.83

1.94

±0.52

2.10

2.29

±0.81

1.90

2.59

±2.06

1.85

1.84

±0.33

1.59

1.60

±0.13

1.73

1.91

±0.71

Median

Mean

+SD

Crea

mmol/L

31.40

32.21

±3.32

34.95

34.65

±4.03

30.85

31.05

±2.68

31.85

34.19

±6.38

33.85

34.10

±3.97

33.55

33.17

±2.34

Median

Mean

+SD

BUN

mmol/L

7.15

7.30

±1.31

7.16

7.49

±1.34

6.57

6.55

±0.55

8.07

7.83

±1.19

6.84

6.84

±0.52

6.26

6.75

±1.39

Median

Mean

+SD

ALB

g/L

44.50

45.18

±2.91

45.40

45.46

±1.68

44.17

43.87

±1.35

43.68

43.19

±2.63

41.94

42.32

±1.97

43.95

43.61

±1.62

Median

Mean

+SD

T-BIL

mmol/L

2.99

2.92

±0.22

2.23

2.30

±0.41

2.24

2.25

±0.35

2.67

2.65

±0.39

2.46

2.64

±0.70

2.90

2.92

±0.23

Median

Mean

+SD

GLU

mmol/L

5.04

5.16

±0.44

4.74

5.04

±0.89

4.90

4.87

±0.48

4.75

5.04

±0.85

5.14

5.05

±0.49

5.66

5.71

±0.49

Median

Mean

+SD

Ca

mmol/L

2.68

2.67

±0.09

2.67

2.66

±0.08

2.64

2.64

±0.05

2.62

2.62

±0.07

2.84

2.84

±0.05

2.80

2.83

±0.08

Median

Mean

+SD

IP

mmol/L

2.10

2.08

±0.17

1.93

1.92

±0.17

1.95

1.93

±0.14

2.09

2.15

±0.36

1.70

1.71

±0.16

1.63

1.62

±0.27

Median

Mean

+SD

CHE

mkat/L

34.83

34.50

±5.21

34.54

36.19

±5.28

30.39

32.11

±5.56

35.74

35.06

±5.87

31.97

34.52

±6.78

32.06

33.06

±5.91

Median

Mean

+SD

BA

mmol/L

74.89

85.59

±40.83

43.97

47.87

±18.31

58.51

61.95

±33.41

55.89

65.20

±28.66

57.12

60.35

±37.38

52.70

58.75

±25.75

Median

Mean

+SD

LDL Direct

mmol/L

0.46

0.52

±0.16

0.41

0.41

±0.08

0.45

0.47

±0.14

0.45

0.47

±0.16

0.43

0.43

±0.04

0.42

0.43

±0.03

Median

Mean

+SD

HDL Direct

mmol/L

0.54

0.57

±0.13

0.52

0.50

±0.05

0.53

0.53

±0.08

0.60

0.57

±0.11

0.55

0.54

±0.02

0.54

0.54

±0.05

Median

Mean

+SD

 

Na

 

mmol/L

140.00

140.60

±1.51

144.50

144.20*

±1.40

142.00

141.50

±1.27

142.00

141.50

±1.65

139.00

139.17

±1.33

140.00

139.83

±1.33

Median

Mean

+SD

 

K

 

mmol/L

3.45

3.39

±0.17

3.20

3.21

±0.10

3.30

3.27

±0.21

3.15

3.23

±0.29

3.40

3.37

±0.10

3.40

3.35

±0.08

Median

Mean

+SD

 

Cl

 

mmol/L

105.00

104.40

±2.41

105.00

105.30

±1.70

107.00

106.20

±1.69

105.00

105.60

±1.71

103.50

103.17

±2.04

107.00

106.17

±1.83

Median

Mean

+SD

 

Note:grey field= values statistically significant (p<0.05)

         *value out of the historical control range

 

Table 23: Examination of thyroid hormones males - T4, T3, TSH hormones

(mean concentration)

Hormone

0

125

500

1000

0 S

1000 S

T4(µg%)

5.154

±

0.738

4.976

±

0.528

5.021

±

0.476

4.830

±

1.150

5.700

±

0.666

4.670

±

0.641

T3(ng/mL)

0.812

±

0.093

0.795

±

0.061

0.745

±

0.093

0.799

±

0.088

0.876

±

0.095

0.806

±

0.096

TSH(pg/mL)

1439.6

±

469.0

1271.2

±

315.5

1493.7

±

381.9

1103.7

±

353.0

1399.1

±

405.4

1311.3

±

485.4

 

Note:grey field= values statistically significant (p<0.05)

 

Table 24: Examination of thyroid hormones females - T4, T3, TSH hormones

(mean concentration)

Hormone

0

125

500

1000

0 S

1000 S

T4(µg%)

4.077

±

0.757

4.215

±

0.869

4.227

±

0.433

4.350

±

0.654

3.960

±

0.997

3.642

±

0.586

T3(ng/mL)

0.750

±

0.185

0.713

±

0.100

0.714

±

0.086

0.696

±

0.127

0.739

±

0.035

0.726

±

0.037

TSH(pg/mL)

1518.8

±

476.2

1021.4

±

152.7

1201.6

±

345.7

970.5

±

201.3

1164.1

±

367.8

1080.2

±

374.5

 

Note:grey field= values statistically significant (P < 0.05)

 

Table 25: Urinalysis males

Parameter

Value

0

125*

500

1000

0 S

1000 S

Volumem/L

mean

8.40

10.11

9.40

6.90

11.67

8.33

Colour(subjective)

num

light

yellow - 9

yellow - 1

light

yellow - 9

 

light

yellow - 8

yellow - 2

light

 yellow - 9

yellow - 1

light yellow - 5

dark yellow - 1

light

 yellow - 6

Cloud(subjective)

num

0-10

0-9

0-10

0-10

0-6

0-6

Odour(subjective)

num

0-10

0-9

0-10

0-10

0-6

0-6

Glucosemg/dL

num

0-10

0-9

0-10

0-10

0-5

50-1

0-6

Proteinmg/dL

num

0-9

15-1

0-6

15-2

30-1

0-6

15-3

30-1

0-4

15-5

100-1

 

0-4

15-1

30-1

0-3

15-2

30-1

Bilirubinmg/dL

num

0-10

0-9

0-10

0-10

0-6

0-6

Urobilinogenmg/dL

num

0-10

0-9

0-10

0-10

0-6

0-6

pH

mean

8.05

8.06

7.90

8.00

8.00

8.17

Specific gravity

 

< 1.005 – 9

1.010 – 1

< 1.005 – 7

1.010 – 2

< 1.005 – 4

1.010 – 4

1.015 - 2

1.010 – 5

1.015 - 4

1.020 - 1

 

< 1.005-5

1.010-1

 

< 1.005 – 5

1.010 - 1

Bloodmg/dL

num

0-8

0.03-2

0-8

0.03-1

0-10

0-10

 

0-6

 

0-6

Ketonesmg/dL

num

0-10

0-9

0-10

0-10

0-6

0-6

Nitrite

num

0-10

0-9

0-10

0-10

0-6

0-6

LeukocytesLeu/μL

num

0-5

75-2

250-1

500-2

0-4

25-1

250-2

500-2

0-5

75-1

250-1

500-3

0-3

75-1

250-2

500-4

0-2

25-2

500-2

0 – 3

25 – 1

500 – 2

 

Note:0 - 0 = measured value - number of animals with changed parameters

         *male No. 23 – died during the study – examination of urine was not performed

 

Table 26: Urinalysis females

Parameter

Value

0

125

500

1000

0 S

1000 S

Volumem/L

mean

5.50

8.00

5.40

6.80

6.50

8.00

Coloursubjective

num

light

yellow - 10

light

 yellow - 10

 

light

yellow - 7

yellow -2

dark yellow -1

light

yellow - 7

yellow - 3

light

 yellow - 6

light

 yellow - 6

Cloudsubjective

num

0- 10

0-10

0-10

0-10

0-6

0-6

Odoursubjective

num

0-10

0-10

0-10

0-10

0-6

0-6

Glucosemg/dL

num

0-10

0-10

0-10

0-10

0-6

0-6

Proteinmg/dL

num

0-10

0-9

15-1

0-10

0-9

15-1

0-5

30-1

0-6

Bilirubinmg/dL

num

0-10

0-10

0-10

0-10

0-6

0-6

Urobilinogenmg/dL

num

0-10

0-10

0-10

0-10

0-6

0-6

pH

mean

7.65

8.35

8.70

8.65

8.00

8.00

Specific gravity

 

< 1.005 – 10

< 1.005 – 10

< 1.005 – 5

1.010- 3

1.015-1

1.020-1

1.010-2

1.015-4

1.020-4

 

< 1.005 – 5

1.010-1

 

< 1.005 – 4

1.010-1

1.015-1

Bloodmg/dL

num

0-9

0.03 -1

0-10

0-9

1-1

0-10

0-6

0-6

Ketonesmg/dL

num

0-10

0-10

0-10

0-10

0-6

0-6

Nitrite

num

0-10

0-10

0-10

0-10

0-6

0-6

LeukocytesLeu/μL

num

0-10

0-9

250-1

0-10

 

0-8

25-1

500-1

0-5

500-1

 

0-4

75-1

500-1

 

Note:0 – 0 = measured value - number of animals with changed parameters

 

Table 27: Males weight of organs (absolute)group mean ± SD (grams)

Organ

0

125

500

1000

0 S

1000 S

Thymus

0.3002

±

0.0596

0.3286

±

0.0678

0.3214

±

0.0864

0.3309

±

0.0667

0.3360

±

0.0613

0.2585

±

0.0431

Adrenal glands

0.0698

±

0.0075

0.0761

±

0.0066

0.0662

±

0.0079

0.0675

±

0.0094

0.0669

±

0.0067

0.0661

±

0.0105

Brain

2.1446

±

0.0842

2.1143

±

0.1166

2.1387

±

0.1329

2.1270

±

0.0676

2.1520

±

0.1116

2.1953

±

0.0782

Spleen

0.8119

±

0.0865

0.8345

±

0.0842

0.8422

±

0.0993

0.8378

±

0.1087

0.8307

±

0.0854

0.8971

±

0.0678

Heart

1.3994

±

0.1068

1.3441

±

0.1152

1.3545

±

0.0988

1.3236

±

0.1048

1.5374

±

0.1970

1.4649

±

0.0933

Testes

4.4434

±

1.1957

3.8204

±

0.4765

4.2233

±

0.3866

4.4580

±

0.3184

4.2204

±

0.3256

4.0016

±

0.2091

Epididymides

1.6515

±

0.1605

1.5236

±

0.1580

1.6242

±

0.0926

1.6385

±

0.1686

1.6459

±

0.1549

1.6212

±

0.0804

Prostate gland

4.3439

±

0.3756

3.9649

±

0.4964

4.0290

±

0.4108

3.8884

±

0.2813

4.4206

±

0.5931

4.0797

±

0.4389

Kidneys

3.2895

±

0.3532

3.3066

±

0.2579

3.1310

±

0.3825

3.0283

±

0.2066

3.3793

±

0.2906

3.3924

±

0.1536

Liver

13.2750

±

1.3268

14.1689

±

1.8491

12.9860

±

2.0877

12.6441

±

1.3126

14.1006

±

1.7453

13.7304

±

1.9362

Pituitary gland

0.0136

±

0.0020

0.0113

±

0.0028

0.0124

±

0.0036

0.0126

±

0.0028

0.0135

±

0.0019

0.0132

±

0.0015

Thyroid gland

0.0336

±

0.0012

0.0325

±

0.0013

0.0330

±

0.0012

0.0332

±

0.0018

0.0337

±

0.0020

0.0334

±

0.0011

 Body weight

503.16

±

21.81

486.48

±

38.36

492.67

±

34.33

474.77

±

33.49

515.86

±

38.17

520.88

±

42.90

 

Note:grey field= values statistically significant (P < 0.05)

 

Table 28: Females weight of organs (absolute)group mean ± SD (grams)

Organ

0

125

500

1000

0 S

1000 S

Thymus

0.2619

±

0.0401

0.2635

±

0.0554

0.2937

±

0.0559

0.2575

±

0.0542

0.2592

±

0.0221

0.2735

±

0.0550

Adrenal glands

0,0878

±

0.0104

0.0975

±

0.0187

0.0882

±

0.0122

0.0847

±

0.0093

0.0958

±

0.0115

0.1034

±

0.0197

Brain

1.9389

±

0.1098

1.9843

±

0.0643

1.9770

±

0.0688

2.0576

±

0.1232

2.0425

±

0.0592

1.9745

±

0.0648

Spleen

0.5264

±

0.0609

0.5507

±

0.0895

0.5491

±

0.0559

0.5428

±

0.0551

0.5548

±

0.1036

0.5120

±

0.0547

Heart

0.9386

±

0.1011

0.9268

±

0.0706

0.9128

±

0.0719

0.9555

±

0.0670

1.0119

±

0.0984

0.9913

±

0.1328

Ovaries

0.1221

±

0.0168

0.1226

±

0.0186

0.1204

±

0.0197

0.2402

±

0.3678

0.1193

±

0.0151

0.1403

±

0.0472

Uterus

0.8577

±

0.3545

0.9587

±

0.4134

1.0370

±

0.2634

1.0829

±

0.6058

0.8894

±

0.2919

0.8777

±

0.3313

Kidneys

1.7633

±

0.1305

1.8817

±

0.2290

1.7930

±

0.1516

1.9301

±

0.2282

1.8966

±

0.1363

1.9514

±

0.1693

Liver

8.3566

±

1.5637

8.1754

±

1.2754

7.9821

±

0.9803

8.9706

±

1.3849

7.8342

±

0.9288

8.2093

±

1.1261

Pituitary gland

0.0134

±

0.0028

0.0151

±

0.0019

0.0138

±

0.0015

0.0150

±

0.0032

0.0149

±

0.0024

0.0159

±

0.0014

Thyroid gland

0.0304

±

0.0028

0.0305

±

0.0011

0.0310

±

0.0012

0.0315

±

0.0021

0.0315

±

0.0017

0.0311

±

0.0017

 Body weight

274.09

±

12.95

281.82

±

31.28

282.14

±

13.95

282.07

±

9.56

284.39

±

10.22

299.67

±

21.42

 

Note:grey field= values statistically significant (P < 0.05)

 

Table 29: Males weight of organs (relative)group mean ± SD(somatic index, %)

Organ

0

125

500

1000

0 S

1000 S

Thymus

0.0596

±

0.0113

0.0671

±

0.0103

0.0646

±

0.0149

0.0698

±

0.0136

0.0653

±

0.0128

0.0494

±

0.0046

Adrenal glands

0.0139

±

0.0014

0.0157

±

0.0016

0.0135

±

0.0017

0.0143

±

0.0023

0.0131

±

0.0019

0.0128

±

0.0026

Brain

0.4269

±

0.0245

0.4373

±

0.0454

0.4354

±

0.0326

0.4494

±

0.0245

0.4180

±

0.0184

0.4234

±

0.0314

Spleen

0.1613

±

0.0155

0.1726

±

0.0233

0.1713

±

0.0207

0.1761

±

0.0150

0.1614

±

0.0174

0.1726

±

0.0118

Heart

0.2779

±

0.0137

0.2769

±

0.0215

0.2751

±

0.0128

0.2795

±

0.0236

0.2977

±

0.0254

0.2820

±

0.0181

Testes

0.8801

±

0.2085

0.7882

±

0.1063

0.8622

±

0.1133

0.9420

±

0.0792

0.8216

±

0.0865

0.7718

±

0.0637

Epididymides

0.3285

±

0.0311

0.3149

±

0.0407

0.3318

±

0.0372

0.3469

±

0.0444

0.3194

±

0.0252

0.3126

±

0.0234

Prostate gland

0.8649

±

0.0853

0.8183

±

0.1109

0.8210

±

0.1011

0.8246

±

0.0981

0.8569

±

0.0914

0.7829

±

0.0489

Kidneys

0.6526

±

0.0482

0.6832

±

0.0714

0.6349

±

0.0583

0.6390

±

0.0400

0.6566

±

0.0542

0.6536

±

0.0410

Liver

2.6353

±

0.1989

2.9037

±

0.2009

2.6257

±

0.2880

2.6609

±

0.1527

2.7318

±

0.2371

2.6277

±

0.1807

Pituitary gland

0.0027

±

0.0004

0.0023

±

0.0006

0.0025

±

0.0007

0.0027

±

0.0006

0.0026

±

0.0004

0.0025

±

0.0003

Thyroid gland

0.0067

±

0.0003

0.0067

±

0.0008

0.0067

±

0.0005

0.0070

±

0.0007

0.0066

±

0.0008

0.0064

±

0.0005

 

Note:grey field= values statistically significant (P < 0.05)

 

Table 30: Females weight of organs (relative)group mean ± SD (somatic index, %)

Organ

0

125

500

1000

0 S

1000 S

Thymus

0.0955

±

0.0138

0.0937

±

0.0188

0.1046

±

0.0215

0.0910

±

0.0169

0.0912

±

0.0088

0.0913

±

0.0165

Adrenal glands

0.0321

±

0.0039

0.0344

±

0.0033

0.0314

±

0.0047

0.0300

±

0.0031

0.0337

±

0.0041

0.0346

±

0.0063

Brain

0.7082

±

0.0433

0.7105

±

0.0676

0.7019

±

0.0367

0.7305

±

0.0534

0.7188

±

0.0286

0.6613

±

0.0448

Spleen

0.1921

±

0.0214

0.1963

±

0.0322

0.1946

±

0.0166

0.1925

±

0.0190

0.1947

±

0.0333

0.1712

±

0.0171

Heart

0.3431

±

0.0394

0.3303

±

0.0194

0.3237

±

0.0229

0.3389

±

0.0230

0.3555

±

0.0289

0.3298

±

0.0245

Ovaries

0.0445

±

0.0048

0.0439

±

0.0076

0.0426

±

0.0065

0.0837

±

0.1251

0.0419

±

0.0045

0.0469

±

0.0151

Uterus

0.3140

±

0.1330

0.3427

±

0.1512

0.3691

±

0.0987

0.3870

±

0.2220

0.3145

±

0.1093

0.29842

±

0.1294

Kidneys

0.6438

±

0.0455

0.6698

±

0.0646

0.6365

±

0.0583

0.6844

±

0.0787

0.6674

±

0.0499

0.6570

±

0.0275

Liver

3.0486

±

0.5521

2.9006

±

0.3011

2.8251

±

0.2712

3.1778

±

0.4536

2.7526

±

0.2907

2.7316

±

0.2303

Pituitary gland

0.0049

±

0.0010

0.0054

±

0.0005

0.0049

±

0.0005

0.0053

±

0.0010

0.0053

±

0.0009

0.0053

±

0.0006

Thyroid gland

0.0111

±

0.0011

0.0109

±

0.0013

0.0110

±

0.0008

0.0112

±

0.0007

0.0111

±

0.0008

0.0104

±

0.0010

 

Note:grey field= values statistically significant (P < 0.05)

 

Table 31: Macroscopic Findings Males

Parameter/Organ/Finding

0

125

500

1000

0 S

1000 S

Number of examined animals

10

9

10

10

6

6

Number of dead animals (mortality)

0

1*

0

0

0

0

Without pathological findings

9

7

10

10

6

6

Testes and epididymis: marked reduced

1

2

0

0

0

0

 

Note:* male No. 23 died, macroscopic examination was not included in the number of examined males

 

Table 32: Macroscopic findings females

Parameter/Organ/Finding

0

125

500

1000

0 S

1000 S

Number of examined animals

10

10

10

10

6

6

Number of dead animals (mortality)

0

0

0

0

0

0

Without pathological findings

10

10

10

10

6

6

Uterus:dilatation (non-pathological)

2

3

1

3

1

2

 

Table 33: Histopathological findings males

Parameter

Organ/Diagnosis

0

125

500

1000

0 S

1000 S

Number of examined animals

10

2

0

10

6

6

Number of dead animals/Mortality

0

1

0

0

0

0

Without findings

4

0

-

2

0

1

Adrenal glands: cortical vacuolation

0

/

/

0

0

1

Adrenal glands: ectopy of cortex

0

/

/

1

0

0

Heart: focal cardiomyopathy

0

/

/

1

1

1

Heart: focal chondrophication of the heart base

0

/

/

2

0

1

Hypophysis: sporadic cyst

0

/

/

1

0

0

Kidneys: acute hyperemia

0

/

/

1

0

0

Lymph node: sinus dilatation

0

/

/

0

0

1

Lungs: acute alveolar hemorrhage**

2

/

/

2

0

0

Lungs: focal interstitial pneumonia

0

/

/

1

0

0

Liver: hyperplasia and inflammation of the bile ducts

0

/

/

1

0

0

Liver: focal polymorphonuclear hepatitis/necrotic hepatitis

0

/

/

1

1

1

Stomach: dilatation of the basis of gastric glands

1

/

/

0

2

1

Stomach:squamous cyst at the interface of the glandular and cutaneous mucosa

0

/

/

0

0

1

Epididymis: atrophy

0

1

/

0

0

0

Epididymis: oligospermia

0

2

/

0

0

0

Prostate gland: focal atrophy

0

/

/

0

1

1

Testicles: diffuse atrophy/with extinction or degeneration of germinal epithelium

0

2

/

1

0

0

Trachea: dilatation of submucosal glands

0

/

/

1

1

1

Note:* male No. 23 died during the studyat the dose level 125 mg/kg bw/day HMDTMP-H, this death did not related with the test item treatment; histopathological examination was not included in the number of examined males

 **finding related to the animal euthanasia

/ - not investigated, only organs with macroscopic changes were investigated

 

 

Table 34: Histopathological Findings Females

Parameter

Organ/Diagnosis

0

125

500

1000

0 S

1000 S

Number of examined animals

10

3

1

10

6

6

Number of dead animals/Mortality

0

0

0

0

0

0

Without findings

2

0

0

2

3

1

Adrenal glands: ectopy of the medulla in the cortex

0

/

/

1

0

1

Adrenal glands: angiectasia of the medulla

0

/

/

1

1

0

Heart: focal cardiomyopathy

1

/

/

2

0

0

Kidneys: acute haemorrhage in the cortex

0

/

/

1

0

0

Kidneys: acute hyperaemia of the medulla

0

/

/

0

0

1

Liver: focal mononuclear or polymorphonuclear hepatitis/necrotic hepatitis

1

/

/

2

1

1

Lungs: acute alveolar haemorrhage*

1

/

/

1

0

0

Appendix: dilatation with atrophy of the mucosa

0

/

/

1

0

0

Proventriculus: mucosal hyperplasia

0

/

/

1

0

0

Ovary: vacuolation of the corpora lutea

0

/

/

0

0

1

Ovary: dilatation rete ovary

0

/

/

1

0

0

Uterus: hydrometra

2

3

1

3

1

2

Trachea: dilatation of submucosal glands

0

/

/

0

0

1

Trachea: cartilage rings hypoplasia

0

/

/

1

0

0

Skeletal muscle: degeneration and polymorphonuclear inflammation

0

/

/

1

0

0

Skeletal muscle: focal myopathy with mononuclear infiltration

0

/

/

1

0

0

 

Note:  histopathological examination was not included in the number of examined females

*finding related to the animal euthanasia

/ - not investigated,only organs with macroscopic changes were investigated

 

Applicant's summary and conclusion

Conclusions:
In the 90-day oral repeated dose toxicity study with 28-day recovery period with HMDTMP (4-7K), conducted according to OECD Test Guideline 408 and in compliance with GLP, the concluded NOAEL for systemic toxicity was at least 1000 mg active acid/kg bw/day based on no treatment-related adverse effects up to the highest dose tested of 1000 mg active acid/kg bw/day.

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