Potassium salts of [hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid (4-7 K:1)

Endpoint summary

Description of key information

In the key 90-day oral repeated dose toxicity study with 28-day recovery period with HMDTMP (4-7K) (aqueous solution containing 22.6% w/w active acid), conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for systemic toxicity was at least 1000 mg active acid/kg bw/day based on no treatment-related adverse effects at the highest dose tested of 1000 mg active acid/kg bw/day (Výzkumný ústav organických syntéz a.s., 2021a).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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

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

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

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

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.

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.

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.

Key result

Critical effects observed:

no

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
1^stweek	379.81±16.53	381.16±25.01	380.61±18.62	375.40±17.52	378.75±16.21	378.87±15.49
2^ndweek	407.46±18.92	406.71±24.81	406.35±22.52	398.82±21.43	408.45±19.44	409.57±20.74
3^rdweek	431.50±17.78	432.41±34.72	428.38±24.83	422.43±27.70	432.43±25.47	435.68±23.51
4^thweek	452.16±18.48	451.18±41.69	446.89±28.26	436.33±29.46	451.62±26.43	452.57±28.26
5^thweek	466.45±18.23	453.23±30.64	461.08±30.04	453.85±32.12	465.47±27.06	469.22±33.75
6^thweek	481.10±20.93	465.91±34.13	471.71±32.14	461.18±34.70	481.32±31.33	479.40±37.16
7^thweek	489.98±19.17	477.61±34.47	481.28±32.53	471.25±38.66	490.50±33.88	490.98±37.69
8^thweek	502.73±21.15	487.21±37.16	493.24±35.35	482.89±38.07	499.22±32.38	497.23±37.35
9^thweek	512.08±19.04	498.29±39.05	502.89±33.93	489.89±38.12	506.15±29.64	504.65±40.82
10^thweek	519.08±20.95	504.93±37.20	510.59±36.34	497.09±38.67	515.35±34.36	516.40±42.13
11^thweek	526.88±21.86	512.02±38.92	518.32±37.54	504.41±36.63	522.77±33.87	523.50±43.51
12^thweek	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
14^thweek	-	-	-	-	540.27±34.33	543.22±49.08
15^thweek	-	-	-	-	539.50±36.06	549.70±50.13
16^thweek	-	-	-	-	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:* 13^thweek - 1^stweek of study (satellite: 17^thweek - 1^stweek of study)

 

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

Time interval	0	125	500	1000	0 S	1000 S
1^stweek	5.12	5.03	4.96	4.43	4.75	4.90
2^ndweek	3.95	3.65	3.68	3.35	4.24	4.39
3^rdweek	3.43	3.67	3.15	3.37	3.43	3.73
4^thweek	2.95	2.68	2.64	1.99	2.74	2.41
5^thweek	2.04	0.29	2.03	2.50	1.98	2.38
6^thweek	2.09	1.81	1.52	1.05	2.26	1.45
7^thweek	1.27	1.67	1.37	1.44	1.31	1.65
8^thweek	1.82	1.37	1.71	1.66	1.25	0.89
9^thweek	1.34	1.58	1.38	1.00	0.99	1.06
10^thweek	1.00	0.95	1.10	1.03	1.31	1.68
11^thweek	1.11	1.01	1.10	1.05	1.06	1.01
12^thweek	0.76	1.20	1.29	0.70	1.17	0.63
13^thweek	1.77	1.22	0.75	0.92	1.40	0.93
14^thweek	-	-	-	-	0.13	1.39
15^thweek	-	-	-	-	-0.11	0.93
16^thweek	-	-	-	-	0.45	1.26
17^thweek	-	-	-	-	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
1^stweek	239.64±12.35	243.02±29.27	243.19±9.81	242.36±11.58	238.93±6.08	246.82±10.67
2^ndweek	250.11±11.63	250.63±26.01	253.39±9.95	251.64±10.97	247.32±8.58	259.82±15.85
3^rdweek	254.76±12.13	258.22±28.91	257.75±12.76	258.52±11.40	251.25±8.80	267.57±14.23
4^thweek	265.06±12.90	267.88±27.06	271.60±12.36	267.97±11.22	266.18±9.36	274.48±17.15
5^thweek	269.00±14.82	270.10±30.43	277.94±14.08	279.56±12.65	271.27±6.76	282.72±14.27
6^thweek	274.64±11.40	275.14±26.58	282.61±14.08	281.97±11.65	271.28±6.40	283.20±14.49
7^thweek	277.18±11.83	285.60±28.18	282.90±16.91	284.59±10.82	280.85±13.60	292.32±17.46
8^thweek	286.80±20.00	289.66±32.24	291.13±16.27	290.63±10.07	282.97±11.65	298.07±18.94
9^thweek	289.31±20.62	288.70±34.56	295.86±15.38	291.92±13.30	282.13±9.26	299.88±18.44
10^thweek	292.74±13.71	295.62±33.03	300.25±15.84	298.96±8.05	287.55±11.23	302.05±16.65
11^thweek	296.14±21.32	298.32±32.42	299.14±17.44	302.11±11.98	289.02±12.94	308.55±20.12
12^thweek	297.49±18.75	305.41±33.15	307.94±16.63	307.19±10.17	299.18±10.78	314.43±23.49
13^thweek	300.86±16.65	306.52±34.04	313.17±15.71	309.16±8.52	298.07±8.71	314.15±18.55
14^thweek	-	-	-	-	300.45±11.09	318.18±22.20
15^thweek	-	-	-	-	305.28±11.89	322.32±26.19
16^thweek	-	-	-	-	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:* 13^thweek - 1^stweek of study (satellite: 17^thweek - 1^stweek of study)

 

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

Time interval	0	125	500	1000	0 S	1000 S
1^stweek	1.04	1.48	1.60	1.29	1.08	1.92
2^ndweek	1.50	1.09	1.46	1.33	1.20	1.86
3^rdweek	0.66	1.08	0.62	0.98	0.56	1.11
4^thweek	1.47	1.38	1.98	1.35	2.13	0.99
5^thweek	0.56	0.32	0.91	1.66	0.73	1.18
6^thweek	0.81	0.72	0.67	0.34	0.00	0.07
7^thweek	0.36	1.49	0.04	0.37	1.37	1.30
8^thweek	1.37	0.58	1.18	0.86	0.30	0.82
9^thweek	0.36	-0.14	0.68	0.18	-0.12	0.26
10^thweek	0.49	0.99	0.63	1.01	0.77	0.31
11^thweek	0.49	0.39	-0.16	0.45	0.21	0.93
12^thweek	0.19	1.01	1.26	0.73	1.45	0.84
13^thweek	0.56	0.19	0.87	0.33	-0.19	-0.05
14^thweek	-	-	-	-	0.34	0.58
15^thweek	-	-	-	-	0.69	0.59
16^thweek	-	-	-	-	3.18	-2.30
17^thweek	-	-	-	-	-3.38	2.77

 

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

Time period	0	125	500	1000	0 S	1000 S
1^sttime period	27.67	28.30	26.80	28.69	27.99	27.47
2^ndtime period	26.04	26.76	26.65	25.73	25.94	26.07
3^rdtime period	28.37	28.42	27.47	27.43	28.30	27.95
4^thtime period	28.09	28.53	27.36	26.86	28.11	27.47
5^thtime period	27.71	27.34	26.72	26.92	28.31	27.34
6^thtime period	24.14	23.48	23.36	23.03	24.67	23.60
7^thtime period	31.17	30.80	30.45	28.35	31.29	30.90
8^thtime period	28.24	27.64	26.98	27.16	27.89	27.55
9^thtime period	27.10	26.82	26.31	25.76	27.41	26.36
10^thtime period	27.98	27.20	27.42	27.09	27.53	27.99
11^thtime period	27.35	26.63	26.42	25.89	27.55	26.37
12^thtime period	25.86	26.95	26.04	26.65	27.53	26.64
13^thtime period	-	-	-	-	26.57	28.48
14^thtime period	-	-	-	-	23.69	23.34
15^thtime period	-	-	-	-	29.58	31.04
16^thtime 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
1^sttime period	21.43	20.59	20.86	22.07	22.90	21.71
2^ndtime period	17.98	19.58	19.63	19.09	18.19	18.69
3^rdtime period	12.26	15.06	16.47	12.38	14.53	13.83
4^thtime period	25.27	26.80	26.15	27.31	26.65	26.24
5^thtime period	19.02	18.64	19.05	16.97	17.65	19.58
6^thtime period	15.01	16.80	15.86	17.61	16.28	17.06
7^thtime period	22.06	23.76	23.55	24.19	22.05	23.41
8^thtime period	19.98	22.30	20.09	24.63	21.97	21.54
9^thtime period	18.77	19.20	19.00	16.56	16.20	19.46
10^thtime period	18.64	16.39	21.36	18.75	21.05	20.41
11^thtime period	18.82	20.55	19.33	20.34	19.06	19.98
12^thtime period	17.97	20.13	20.10	19.10	17.67	20.73
13^thtime period	-	-	-	-	19.11	20.62
14^thtime period	-	-	-	-	15.88	17.99
15^thtime period	-	-	-	-	22.30	23.34
16^thtime 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
1^sttime period	14.28	12.90	13.73	11.68	15.15	15.98
2^ndtime period	13.17	13.71	11.82	13.10	13.22	14.31
3^rdtime period	10.40	9.43	9.61	7.25	9.68	8.62
4^thtime period	7.26	1.02	7.42	9.31	7.04	8.66
5^thtime period	7.54	6.62	5.69	3.90	7.98	5.30
6^thtime period	5.26	7.11	5.86	6.25	5.31	6.99
7^thtime period	5.84	4.45	5.62	5.86	3.99	2.88
8^thtime period	4.75	5.72	5.11	3.68	3.55	3.85
9^thtime period	3.69	3.54	4.18	4.00	4.78	6.37
10^thtime period	3.95	3.71	4.01	3.88	3.85	3.61
11^thtime period	2.78	4.51	4.88	2.70	4.25	2.39
12^thtime period	6.47	4.53	2.88	3.45	5.09	3.49
13^thtime period	-	-	-	-	0.49	4.88
14^thtime period	-	-	-	-	-0.46	3.98
15^thtime period	-	-	-	-	1.52	4.06
16^thtime 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
1^sttime period	7.00	5.29	7.00	6.03	5.24	8.57
2^ndtime period	3.67	5.52	3.16	5.13	3.08	5.94
3^rdtime period	11.99	9.16	12.02	10.90	14.66	7.16
4^thtime period	2.22	1.19	3.48	6.08	2.74	4.50
5^thtime period	4.26	3.86	3.52	2.00	0.01	0.36
6^thtime period	2.40	8.87	0.25	2.10	8.42	7.62
7^thtime period	6.21	2.44	5.01	3.56	1.36	3.50
8^thtime period	1.80	-0.63	3.38	0.73	-0.55	1.21
9^thtime period	2.61	5.16	3.32	6.10	4.75	1.59
10^thtime period	2.63	2.38	-0.75	2.40	1.00	4.56
11^thtime period	1.01	4.91	6.52	3.59	7.61	4.20
12^thtime period	3.12	0.94	4.33	1.73	-1.08	-0.24
13^thtime period	-	-	-	-	1.78	2.81
14^thtime period	-	-	-	-	4.35	3.28
15^thtime period	-	-	-	-	14.26	-9.85
16^thtime 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
1^sttime period	61.71	64.00	54.14	60.43	58.81	61.90
2^ndtime period	62.43	61.14	51.29	60.43	63.57	61.67
3^rdtime period	60.29	58.14	50.43	58.86	63.10	60.95
4^thtime period	58.86	59.57	50.71	60.71	67.62	60.71
5^thtime period	62.43	61.27	52.29	59.00	65.48	66.67
6^thtime period	60.86	59.68	50.00	58.43	67.62	59.52
7^thtime period	65.71	67.94	55.57	60.29	64.05	60.00
8^thtime period	60.29	59.68	54.14	59.71	65.71	62.38
9^thtime period	63.86	61.90	49.57	56.71	67.38	62.14
10^thtime period	63.29	60.63	48.71	58.43	64.05	62.86
11^thtime period	61.29	59.05	48.71	54.86	66.43	60.24
12^thtime period	66.86	62.06	51.71	62.86	70.00	63.33
13^thtime period	-	-	-	-	61.19	54.52
14^thtime period	-	-	-	-	64.29	58.10
15^thtime period	-	-	-	-	65.95	59.52
16^thtime 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
1^sttime period	47.73	46.71	46.57	46.00	46.43	48.10
2^ndtime period	45.23	43.57	43.43	44.00	44.05	47.14
3^rdtime period	42.43	41.71	43.86	47.43	44.05	42.86
4^thtime period	41.86	42.71	44.14	45.00	45.57	45.48
5^thtime period	44.00	43.86	43.00	46.00	44.29	47.14
6^thtime period	40.29	45.71	42.86	44.14	42.62	44.29
7^thtime period	42.57	43.00	41.71	42.29	39.29	40.00
8^thtime period	45.86	45.00	42.29	40.43	39.05	47.62
9^thtime period	41.14	43.29	43.00	44.43	41.90	43.81
10^thtime period	40.71	45.86	43.71	44.43	42.86	44.71
11^thtime period	37.71	45.00	43.00	41.14	41.67	45.24
12^thtime period	40.14	48.29	46.57	48.86	44.05	47.62
13^thtime period	-	-	-	-	37.86	43.81
14^thtime period	-	-	-	-	40.00	44.29
15^thtime period	-	-	-	-	41.67	44.05
16^thtime 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
1^stweek	1/10	1/10	1/10	1/10	1/6	1/6
2^ndweek	1/10	1/10	1/10	1/10	1/6	1/6
3^rdweek	1/10	1/10	1/10	1/10	1/6	1/6
4^thweek	1/10	1/10	1/10	1/10	1/6	1/6
5^thweek	1/10	1/9*	1/10	1/10	1/6	1/6
6^thweek	1/10	1/9	1/10	1/10	1/6	1/6
7^thweek	1/10	1/9	1/10	1/10	1/6	1/6
8^thweek	1/10	1/9	1/10	1/10	1/6	1/6
9^thweek	1/10	1/9	1/10	1/10	1/6	1/6
10^thweek	1/10	1/9	1/10	1/10	1/6	1/6
11^thweek	1/10	1/9	1/10	1/10	1/6	1/6
12^thweek	1/10	1/9	1/10	1/10	1/6	1/6
13^thweek	1/10	1/9	1/10	1/10	1/6	1/6
14^thweek	-	-	-	-	1/6	1/6
15^thweek	-	-	-	-	1/6	1/6
16^thweek	-	-	-	-	1/6	1/6
17^thweek	-	-	-	-	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 5^thweek 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
1^stweek	1/10	1/10	1/10	1/10	1/6	1/6
2^ndweek	1/10	1/10	1/10	1/10	1/6	1/6
3^rdweek	1/10	1/10	1/10	1/10	1/6	1/6
4^thweek	1/10	1/10	1/10	1/10	1/6	1/6
5^thweek	1/10	1/10	1/10	1/10	1/6	1/6
6^thweek	1/10	1/10	1/10	1/10	1/6	1/6
7^thweek	1/10	1/10	1/10	1/10	1/6	1/6
8^thweek	1/10	1/10	1/10	1/10	1/6	1/6
9^thweek	1/10	1/10	1/10	1/10	1/6	1/6
10^thweek	1/10	1/10	1/10	1/10	1/6	1/6
11^thweek	1/10	1/10	1/10	1/10	1/6	1/6
12^thweek	1/10	1/10	1/10	1/10	1/6	1/6
13^thweek	1/10	1/10	1/10	1/10	1/6	1/6
14^thweek	-	-	-	-	1/6	1/6
15^thweek	-	-	-	-	1/6	1/6
16^thweek	-	-	-	-	1/6	1/6
17^thweek	-	-	-	-	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
1^stweek	1/10	1/10	1/10	1/10	1/6	1/6
2^ndweek	1/10	1/10	1/10	1/10	1/6	1/6
3^rdweek	1/10	1/10	1/10	1/10	1/6	1/6
4^thweek	1/10	1/10	1/10	1/10	1/6	1/6
5^thweek	1/10	1/9*	1/10	1/10	1/6	1/6
6^thweek	1/10	1/9	1/10	1/10	1/6	1/6
7^thweek	1/10	1/9	1/10	1/10	1/6	1/6
8^thweek	1/10	1/9	1/10	1/10	1/6	1/6
9^thweek	1/10	1/9	1/10	1/10	1/6	1/6
10^thweek	1/10	1/9	1/10	1/10	1/6	1/6
11^thweek	1/10	1/9	1/10	1/10	1/6	1/6
12^thweek	1/10	1/9	1/10	1/10	1/6	1/6
13^thweek	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 5^thweek of application

 

 Table 14: Clinical Observation Females

Time interval	0	125	500	1000	0 S	1000 S
1^stweek	1/10	1/10	1/10	1/10	1/6	1/6
2^ndweek	1/10	1/10	1/10	1/10	1/6	1/6
3^rdweek	1/10	1/10	1/10	1/10	1/6	1/6
4^thweek	1/10	1/10	1/10	1/10	1/6	1/6
5^thweek	1/10	1/10	1/10	1/10	1/6	1/6
6^thweek	1/10	1/10	1/10	1/10	1/6	1/6
7^thweek	1/10	1/10	1/10	1/10	1/6	1/6
8^thweek	1/10	1/10	1/10	1/10	1/6	1/6
9^thweek	1/10	1/10	1/10	1/10	1/6	1/6
10^thweek	1/10	1/10	1/10	1/10	1/6	1/6
11^thweek	1/10	1/10	1/10	1/10	1/6	1/6
12^thweek	1/10	1/10	1/10	1/10	1/6	1/6
13^thweek	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	10^9/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	10^12/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	10^9/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	10^9/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	10^12/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	10^9/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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 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

In a 28-day dose range-finding study with HMDTMP (4-7K) (aqueous solution containing 22.6% w/w active acid), oral gavage administration of 0, 125, 250, 500 and 1000 mg active acid/kg bw/day for 28 days to 6 male and 6 female rats did not result in test substance-related mortality, clinical signs of toxicity, body weight changes, changes in haematological parameters or macroscopic changes during necropsy. On the basis of these results dose levels of 125, 250, 500, 1000 mg active acid/kg bw/day were selected for the main 90-day oral repeated dose toxicity study (Výzkumný ústav organických syntéz a.s., 2020a).

 

In the key 90-day oral repeated dose toxicity study with 28-day recovery period with HMDTMP (4-7K) (aqueous solution containing 22.6% w/w active acid), conducted according to OECD Test Guideline 408 and in compliance with GLP (Výzkumný ústav organických syntéz a.s., 2021a), 10 male and 10 female Wistar rats per group were given daily oral (gavage) administration of HMDTMP (4-7K) at doses of 0 (water), 125, 500 and 1000 mg active acid/kg bw/day for 90 days. The control animals were treated in the same manner, but they were given the vehicle, water, only. The control and high dose group also included additional 6 male and 6 female satellite animals which were maintained for further 28 days after administration ceased to detect persistence or reversibility of effects.

During the 90-day administration period, clinical and health status observations were performed daily. Body weight and food consumption were measured weekly, and the detailed clinical observations were carried out in the same time interval. Water consumption was measured twice per week. Ophthalmologic examination was performed during the first week of application and in the last week of treatment and recovery periods. Functional observations were performed during the last week of treatment and the last week of the recovery period. the. The study was completed by urinalysis, haematological and biochemical analysis (including thyroid hormone), and gross necropsy of animals. The organs selected for weighing and histopathological examination were removed. These parameters (except clinical observation) were also checked for the satellite groups of animals.

Oral administration of the test substance did not cause treatment-related mortality or clinical signs of toxicity. Only one male at 125 mg active acid/kg bw/day was found dead due to an intubation error.

No test material-related effects on the body weight, food consumption and water consumption were recorded during the study. Slight changes in these parameters were without statistical significance and were concluded to be non-adverse and of no toxicological importance.

The haematological examination revealed a test material-related effect on haemocoagulation parameters. The concentration of fibrinogen was statistically significantly decreased in all groups of males and females at the highest and lowest dose levels. Statistically significantly decreased partial thromboplastin time (APTT) was measured in females of the mid dose level and statistically significantly decreased prothrombin time (PT) was noted in males of the mid dose level. These changes were observed in both sexes but were not associated with any pathological or histopathological findings of haematogenous organs. Sporadic changes in the values of red blood components were measured in females: increased total erythrocyte count, haemoglobin concentration, haematocrit (HCT) at the lowest dose level and in females after recovery period. The reticulocyte count was decreased in females at the lowest and highest dose levels and decreased in males at the end of the recovery period. These statistically significant differences were without a dose-response relationship and without a correlation with other manifestations of toxicity.

Biochemical examination revealed statistically significant changes without dose dependence, sex correlation and without association with related toxicologically significant endpoints. In males of the highest dose level, reversibly increased urea and cholinesterase and decreased albumin concentrations were reported. Glucose concentration was decreased in males of the mid dose level at the end of the exposure period. Increased glucose concentration was measured and in females at the highest dose level at the end of the recovery period. In males the concentration of calcium ions was increased in the lowest dose level and decreased at the mid dose level. In females the concentration of sodium ions was increased at the lowest dose level at the end of the treatment period and the concentration of chloride ions was increased at the highest dose level at the end of the recovery period. Potassium ion concentration was decreased only in males at the highest dose level at the end of the exposure period. Total bilirubin was statistically significantly decreased in females at the lowest and mid dose levels at the end of the exposure period. Reversibly increased activity of alkaline phosphatase (ALP) was found only in females of the highest dose level. These differences were without dose response relationship and without correlation with other manifestations of toxicity.

Urine parameters were affected in both sexes, but statistically significant differences were without a dose-response relationship and without a correlation with histopathological findings of urinary tract. In females at the lowest and highest dose level the volume of urine was significantly increased at the end of the treatment period. In males at the highest dose level the volume of urine was statistically significantly decreased at the end of the recovery period. The presence of proteins and leucocytes in urine was detected only in the mid and the highest dose levels. None of the findings were associated with the application of the test material.

Biometry of organs showed several statistically significantly changes in absolute and relative organ weights. In high dose males, a statistically significant increase in absolute and relative testes weight were measured at the end of the exposure period but appeared normal at the end of the recovery period. In high dose recovery males, a statistically significant decrease in absolute and relative thymus weight was recorded at the end of recovery period. A statistically significant increase in relative liver weight was recorded in mid dose group males, which was not observed in any other treatment group or following the recovery period. In treatment group females there were no changes detected in absolute and relative organ weights. In high dose recovery females, a statistically significant decrease in absolute and relative heart weight was observed. Overall, the statistically significant changes in organ weights did not correlate with any histopathological findings and appeared to be of a sporadic and non-adverse origin.

No toxicologically significant findings were detected during the histopathological examination of organs and tissues from animals at the highest dose level and satellite animals. Sporadic findings did not relate to treatment with the test material.

The NOAEL for systemic toxicity was concluded to be 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.

 

Justification for classification or non-classification

Based on the available data, the substance does not require classification for specific target organ toxicity following repeated exposures according to Regulation (EC) No 1272/2008.