Reaction product of disodium 4,4'-dinitrostilbene-2,2'-disulphonate, p-[(4-amino-2,5-xylyl)azo]benzenesulphonic acid, 3-[(4-amino-2-methoxyphenyl)azo]-4-hydroxybenzenesulphonic acid, copper (II) sulfate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.07.2016 – 19.10.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted by the Council on 29th July 2016

Deviations:

yes

Remarks:

see Any other information ...

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 15-[(Z)-2-{4-[(E)-2-{4-[(E)-2-{2,5-dimethyl-4-[(E)-2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]-2-sulfophenyl}ethenyl]-3-sulfophenyl}diazen-1-yl]-10,12-dioxa-2,3-diaza-11-cupratricyclo[11.4.0.0^{4,9}]heptadeca-1(17),2,4,6,8,13,15-heptaene-6-sulfonate

EC Number:

943-210-6

Molecular formula:

Not applicable. UVCB Substance

IUPAC Name:

tetrasodium 15-[(Z)-2-{4-[(E)-2-{4-[(E)-2-{2,5-dimethyl-4-[(E)-2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]-2-sulfophenyl}ethenyl]-3-sulfophenyl}diazen-1-yl]-10,12-dioxa-2,3-diaza-11-cupratricyclo[11.4.0.0^{4,9}]heptadeca-1(17),2,4,6,8,13,15-heptaene-6-sulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: by Sponsor, Batch No. 8004/2008- Expiration date of the lot/batch: 02/2018- Storage condition of test material: The test substance was stored in dry and dark place at room temperature- Stability under test conditions:From the results of analyses it follows that the both application forms (10 mg and 1000 mg/10 mL) of the test substance, Direct Brown 115, Direct Brown 116, prepared at defined laboratory conditions (laboratory temperature, sonication, stirring) are homogenous and stable at least for 120 minutes from the finalization of application form preparation.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: see below Details on oral exposureFORM AS APPLIED IN THE TEST (if different from that of starting material)solution in water for injection

Test animals

Species:

rat

Strain:

Wistar

Remarks:

CRL (SPF quality - guaranteed)

Details on species / strain selection:

- according to guidelinerandom selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex did not exceed ± 20% of the mean weight

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., Czech Republic, RČH CZ 11760500- Females (if applicable) nulliparous and non-pregnant: yes- Age at study initiation: sexually adult, 7-9 weeks on arrival- Weight at study initiation: males 401 - 428 g, females 252 - 264 g- Fasting period before study: no- Housing: SPF conditions according to internal SOP No.12; sterilized soft wood fibers Lignocel;Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – 1 M + 1 F in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage;- Diet (e.g. ad libitum): complete pelleted diet for rats and mice in SPF breeding - Altromin for Rats/Mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany- Water (e.g. ad libitum): ad libitum; quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law- Acclimation period: 12 days (DRFE 5 days)ENVIRONMENTAL CONDITIONS- Temperature (°C): 22±3- Humidity (%): 30-70- Air changes (per hr): approximately 15 air changes per hour- Photoperiod (hrs dark / hrs light): 12 /12STUDY TIME SCHEDULEAdministration (from 04.10.2016)Parental males (totally 49 days of administration):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day ( mating ) → 43rd day – 63rd day (administration period) → 64th day (necropsy) Satellite males (totally 49 days of administration + 14 days of observation):1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 64th day - 77th day (observation period) → 78th day (necropsy)Parental females:1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day (mating)→ gestation → lactation → day 12 post partum Satellite females (totally 49 days of administration + 14 days of observation):1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 64th day - 77th day (observation period) → 78th day (necropsy)Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day (mating) → 25 days after the end of mating period Non-pregnant females (with evidence of copulation):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day (mating) → 25th day after confirmed matingObservationsUrinalysis: only males – 63rd and 77th day of studyBlood collection for haematology and biochemistry: parental males – 64th day of study satellite males – 78th day of studyparental females - 13th day of lactation periodpups – 2 pups per litter – 4th day of lactation; 2 pups per litter – 13th day of lactationsatellite females – 78th day of studyNecropsy: parental males – 64th day of studysatellite males – 78th day of studyparental females - 13th day of lactation periodpups – 2 pups per litter – 4th day of lactation, other pups - 13th day of lactationsatellite females – 78th day of studynon-pregnant females – 26th day after the end of mating period or confirmed matingEnd of histopathological examination: 19.10.2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:The test substance was weighted into glass beaker and the beaker was replenished by water for injections. The test solution was dissolved in ultrasonic bath for a 5 minutes and then the solution was stirred by magnetic stirrer (450 rpm) for 10 minutes. The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. For each dose level concentration, the solution was prepared separately. The application forms were prepared daily just before administration. The administration of the test substance to animals was performed during one hour after preparation of application form. The stirring of solutions continued during administration.VEHICLEwater for injection - Concentration in vehicle:The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Stability and homogeneity were determined by means of measuring of a peak area of the test substance by a liquid chromatography based on a method developed at the test facility.

Duration of treatment / exposure:

The treated groups were administered daily for the following periods: males and females – 2 weeks prior to the mating period and during the mating periodpregnant females – during pregnancy and till the 12th day of lactationmales – after mating period – totally for 49 daysnonpregnant females (mated females without parturition) – for 25 days after the confirmed matingnon-mated females – for 25 days after the end of mating period

Frequency of treatment:

7 days per week at the same time

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

(vehicle only)

No. of animals per sex per dose:

12 females and 12 males per group, 6 males and 6 females per satellite group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for study were determined on the basis of results of a dose-range finding experiment. - Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

HEALTH CONDITION CONTROL: Yes - Time schedule: daily - during the acclimatization and the experimental partMORTALITY CONTROL: - Time schedule: twice dailyCLINICAL OBSERVATIONS: Yes males and females - daily during the administration period in natural conditions in cagesDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: before the first application and then weekly (except the mating period)FUNCTIONAL OBSERVATIONS: Yes - Time schedule: at the end of administration / observation periodNEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: at the end of administration / observation period- Dose groups that were examined: 6 males and 6 females of each group and in satellite males and females- Battery of functions tested: sensory activity / grip strength / motor activityBODY WEIGHT: Yes- Time schedule for examinations:males - the first day of administration and then weekly,females - the first day of administration and then weekly in premating and mating period, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 1st, 4th, 12th and 13th day,satellite males and females - the first day of administration and then weekly.FOOD CONSUMPTION: - Food consumption determined and group mean daily diet consumption calculated as g food/animal/day: Yes- Time schedule: weekly and on the same days as body weight (except the mating period); satellite males and females – weeklyFOOD EFFICIENCY:- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: YesTime schedule: males - weekly (except the mating period), females - weekly during premating period, during pregnancy and lactation – on the same days as body weight, satellite males and females – weeklyWATER CONSUMPTION: Yes- Time schedule for examinations: twice a week (only in satellite animals)HAEMATOLOGY: Yes- Time schedule for collection of blood: at the end of administration (M) / observation period (satellite M,F)- Anaesthetic used for blood collection: Yes, light ether narcosis- Animals fasted: Yes- How many animals: 6 males and 6 females of each group and in satellite males and females- Parameters checked in table [No.3; tables in the attached document] were examined.CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: at the end of administration (M, non-pregnant F) / observation period (satellite M,F)- Animals fasted: Yes- How many animals: 6 males and 6 females of each group and in satellite males and females- Parameters checked in table [No.4] were examined.URINALYSIS: Yes- Time schedule for collection of urine: the last day of administration / observation period – only males- Metabolism cages used for collection of urine: Yes- Animals fasted: Not specified- Parameters checked in table [No.2] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities and biometry of organsHISTOPATHOLOGY: Yes (see table [No.5] Organs for histopathological examination). In Repeated Dose Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs with macroscopical changes and rectum, skeletal muscle, intestines, testes, epididymis were examined at the lowest and middle dose level groups used for Repeated Dose Toxicity part of study.

Statistics:

For statistical evaluation the software Statgraphic ® Centurion (version XVII, USA) was used. Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group. The results statistically significant on probability level 0.05 were indicated in the summary tables in report.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Males/Females: Only changes related to the colour of the test substance – coloured excrements and urine were recorded at the dose level 500 and 1000 mg/kg/day.In one female at the dose level 1000 mg/kg/day hunched posture and piloerection were recorded during the study. M/F:The activity (poise, gait, reaction to handling) of all males of all treated groups was similar during the study and not different from the activity of males of the control group.At the 2nd week of application period vocalisation in some females at the dose level 1000 mg/kg/day was detected.

Mortality:

no mortality observed

Description (incidence):

No mortality treatment related was observed in main groups.Female No.193 (at the satellite treated group) died on the 42nd day of application. This death probably relates to the test substance application.Female No.162 (at the dose level 1000 mg/kg/day) died on the 14th day of application due to intubation error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly decreased mean of body weight was detected in animals of both sexes at the highest dose level. The body weight increment in treated animals was decreased or variable. In some time weight period the body weight gain was decreased by more than 10% compared to control so this effect could be regarded as toxicologically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of males at the highest dose level 1000 mg/kg/day was slightly lower or comparable to the control males for the whole time of application period.

Food efficiency:

no effects observed

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

not examined

Description (incidence and severity):

only satelite groups

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological examination of males was without changes and females showed sporadic findings only; all findings were without dose dependence. Only differential percentage counts of lymphocytes and granulocytes were significantly changed without dose dependency at the dose level 500 mg/kg/day. The concentration of fibrinogen (FIB) was statistically significantly increased in the satellite treated females. All findings observed in both sexes (but in absence of a treatment-related clinical signs of toxicity) were considered to be of no toxicological significance.All haematological parameters were in range of historical control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical examination showed increased value of protein total and albumin in both sexes (irreversible in females). Reversibly increased relative weight of liver and kidneys were detected in males and females. This probably related to the adaptation process of organism to the test substance administration. Increased activity of hepatic enzymes could be also related with metabolism of test substance in organism. For complete elimination of these symptoms (increased value of protein total and albumin) be probably needed to longer time of recovery period. Biochemical examination showed decreased value of creatinine in males in all treated groups and in females at the highest dose level. Also increased activity of AST and ALT in males and females was detected. These values (values of creatinine, values of activity of AST and ALT) were out of historical control limits. Decrease of these parameters related to serious damage of muscular matter which was confirmed during the functional observation and by the histopathological examination.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly decreased volume of urine was recorded in all male treated groups (dose dependent) in the dose levels 0-250-500-1000 Change of colour of urine (yellow or dark orange) in 0-2-6-4 males was recorded. Higher specific weight of urine in two males at the dose level 1000 mg/kg/day was detected. Presence of proteins in 0-2-3-2 males, presence of blood in 0-0-3-1 males, presence of ketones in 0-0-0-3 males and presence of nitrite in 0-0-3-0 males were found out. Presence of leucocytes in 2-2-2-2 males was recorded.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

During the functional observation markedly decreased number of upstanding in males at the middle and highest dose levels and in females at the highest dose level was recorded. Biochemical examination showed decreased value of creatinine in males in all treated groups and in females at the highest dose level. Also increased activity of AST and ALT in males and females was detected. These values (values of creatinine, values of activity of AST and ALT) were out of historical control limits. Decrease of these parameters related to serious damage of muscular matter which was confirmed during the functional observation and by the histopathological examination.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Reversibly increased relative weight of liver and kidneys were detected in males and females. This probably related to the adaptation process of organism to the test substance administration. Increased activity of hepatic enzymes could be also related with metabolism of test substance in organism. For complete elimination of these symptoms (increased value of protein total and albumin) be probably needed to longer time of recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Males: Dark orange colour of urine was recorded in 0-0-0-3 males. In epididymis reduction and/or grey-blue colour in 0-0-0-3 in males were observed. Reduced testes of flabby consistency in 0-0-0-3 males were found out.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination showed findings in rectum (infiltration of mucosa and/or submucosa, brown pigment in mucosa and focal erosion) and intestines (brown pigment in mucosa, epithelium coloured by the test substance). Occurrence of microscopic findings in rectum and/or intestine probably related with place of application (digestive tract). Presence of brown pigment in mucosa in rectum did not disappear even after the end of recovery period. During the histopathological examination also serious finding on skeletal muscle in males and females (atrophy of muscle fibres) was found out.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

other: determined on the basis results of histopathological examinations and biochemical examinations

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Deviations:

During the study, short increase of temperature (31.03.2017 - for ten hours, temperature from 25.1 to 25.9°C and 01.04. 2017 for thirteen hours, temperature from 25.4 to 26.7°C) in animal room was recorded. This microclimatic deviation did not affect the welfare of animals and had no impact on the results of the study.

One deviation from the Study Plan occurred. The study was performed according to the updated OECD Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on the 29th July 2016 instead of version given in Study Plan. The differences between these two versions are not substantial for performance of study (some corrections in number of pups for T4 examination, changes in list organs to weight determination, calculation of fertility parameters etc.). In this study, these changes were respected.

No other deviations from study plan or test guidelines were observed during the study performance.

Applicant's summary and conclusion

Conclusions:

The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 250 mg/kg body weight/day both for MALES and FEMALES. The value of NOAEL was determined on the basis results of histopathological examinations and biochemical examinations.

Executive summary:

Introduction

The test substance, Reaction products of 3-amino-4-hydroxybenzenesulfonic acid, 3-aminophenol, (E)-6,6'-(ethene-1,2-diyl)bis(3-nitrobenzenesulfonic acid), 2,5-dimethylbenzenamine, 4-aminobenzenesulfonic acid, chelated with copper, sodium salt, was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on 28th July 2015.

 Further in the text the simplified name is used for the test substance, i.e. “Direct Brown 115, Direct Brown 116”, instead of “Reaction products of 3-amino-4-hydroxybenzenesulfonic acid, 3-aminophenol, (E)-6,6'-(ethene-1,2-diyl)bis(3-nitrobenzenesulfonic acid), 2,5-dimethylbenzenamine, 4-aminobenzenesulfonic acid, chelated with copper, sodium salt.”

 Methods

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 250, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment (see the Annex 2) and approved by Sponsor.

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females – during pregnancy and till the 12th day of lactation,

males – after mating period – totally for 49 days,

nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating,

non-mated females – for 25 days after the end of mating period

After the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.   

During the study clinical observation and health status controls were performed daily. The body weight and food consumption were measured weekly. Detailed clinical observation was carried out weekly. The functional observation was performed at the end of application and observation period. Vaginal smears were prepared daily, 2 weeks before start of administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy day. Reproduction parameters relevant to pups (number of pups, weight of litters, weight, sex and vitality of pups, measurement of anogenital distance, nipple retention) were also recorded.

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of main groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from parental animals and pups were removed for weighing and histopathological examination.

 

Results

Repeated Dose Toxicity part of study:

At the dose level 250 mg/kg/day – no mortality of animals were recorded. No changes during health condition control and clinical observation were observed.

Body weight, body weight increments, food consumption and water consumption of treated animals were not significantly affected by the test substance administration.

In males significantly increased value of platelet count was recorded during the haematological examination (but in range of historical control).

Significantly increased values of albumin, protein total and activity of AST and significantly decreased values of creatinine and inorganic phosphorus in males were detected during the biochemical examination but all values were in range of historical control.

In females no significant haematological or biochemical parameters were detected.

During examination of urine in males significantly decreased mean of urine volume and presence of protein in two males and change of colour in two males were recorded.

No significant changes of organs were recorded during the biometry of organs, macroscopic examination and histopathological examination.

 

At the dose level 500 mg/kg/day – no mortality of animals were recorded. No changes during health condition control and clinical observation were observed (except coloured excrements and/or urine). During the functional observation the number of upstanding in males was decreased.

Body weight, body weight increments, food consumption and water consumption of treated animals were not significantly affected by the test substance administration.

During the haematological examination significantly increased value of platelet count and prolonged prothrombin time in males were recorded. These parameters were in range of historical control limits.

Significantly increased values of albumin, protein total, calcium ions, cholinesterase, bilirubin total and activity of AST, ALP, AST, and significantly decreased values of creatinine in males and significantly increased values of protein total and albumin in females were detected during the biochemical examination. The values of creatinine and AST in males and value of albumin in females were out of historical control limits.

During examination of urine in males significantly decreased mean of urine volume and presence of protein in three males, presence of blood in two males, presence of nitrite in three males and change of colour in three males were recorded.

Significantly increased relative weight of kidneys and liver in both sexes was recorded during the examination of biometry of organs.

No significant changes of organs were recorded during the macroscopic examination. Only non-pathological finding as coloured content in stomach and intestines was detected but this after end of administration of the test substance disappeared.

During the histopathological examination focal atrophy of muscle fibres in skeletal muscle, infiltration of mucosa and submucosa in rectum in males and brown pigment in mucosa in rectum in both sexes were found out. 

 

At the dose level 1000 mg/kg/day – one female died on the 14th day of application due to intubation error. One satellite female died on the 42nd day of application – this probably related to the test substance treatment. No significant changes during health condition control and clinical observation were observed in both sexes. Only coloured excrements and/or urine were detected during the application period. During the functional observation the number of upstanding in males and females was decreased.

During the study significantly decreased body weight in animals in both sexes was recorded. Body weight increment was decreased in males and in females was variable. Variability of value of mean of food consumption and food conversion was also recorded. 

Haematological examination showed significantly prolonged PT, significantly decreased values of RBC, haemoglobin and haematocrite and delayed significant increase platelet count and delayed shortened APTT in males. In females irreversibly decreased values of haematocrite and haemoglobin, delayed significantly decreased value of fibrinogen were recorded.

 

Significantly increased activity of ALP, ALT, AST (reversible) and value of bilirubin total and decreased value of creatinine and glucose, delayed significant increase value of cholinesterase and delayed significant decrease value of sodium ions in males were recorded during the biochemical examination. In females, irreversibly increased values of protein total and albumin, irreversibly decreased value of creatinine, significantly increased activity of AST, delayed increased values of triglyceride and glucose, delayed significantly decreased value of inorganic phosphorus were detected during the biochemical examination. The values of activity of ALP, ALT, AST, values of creatinine, bilirubin total in males and values of albumin and protein total in females were out of historical control limits. 

During examination of urine in males significantly decreased mean of urine volume was detected. Presence of protein in two males, presence of blood in one male, presence of ketone in three males and change of colour in four males were recorded.

Irreversibly decreased absolute weight of prostate gland+seminal vesicles, testes and epididymis (in testes and epididymis significance delayed), significantly decreased absolute weight of thymus and delayed increased absolute weight of adrenal glands in males were recorded. In females delayed increased absolute weight in liver was recorded during the examination of biometry of organs. Relative weight of kidneys, liver, pituitary gland and brain was significantly changed in males also irreversible decreased relative weight in testes and epididymis (significance delayed) in males and significantly increased relative weight of kidneys, liver and brain, delayed significant decrease relative weight of spleen and ovaries in females were recorded.    

The macroscopic changes of epididymis and testes in males were found out. Also non-pathological findings as coloured content in stomach and intestines was detected but these after the end of administration of the test substance disappeared.

Histopathological examination showed significant findings in intestines (brown pigment in mucosa, epithelium coloured by the test substance), in rectum (brown pigment in mucosa, infiltration of mucosa and/or submucosa), in testes (tubular atrophy, tubular dystrophy, brown pigment in interstitium), in epididymis (germ cell and/or cell debris in lumen tubules, brown pigment in interstitium, oligospermia) in skeletal muscle (focal atrophy of muscle fibres) in males. In females in rectum (brown pigment in mucosa, focal erosion, infiltration of mucosa and/or submucosa), in skeletal muscle (atrophy of muscle fibres), in rectal vein (vessels thrombus, inflammation) and findings in uterus which related to females gravidity or oestrus cycle were found out.  

 

Conclusion

The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 250 mg/kg body weight/day both for MALES and FEMALES. The value of NOAEL was determined on the basis results of histopathological examinations and biochemical examinations.