Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28. 4. – 5. 12. 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
(see Overall remarks section)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ashes (residues) - Biomass Combustion
- Physical state: solid
- Composition of test material, percentage of components:
Complex product of oxides
SiO2 37.81 % (w/w), K2O 17.01 % (w/w), CaO (total) 11.92 % (w/w), P2O5 6.15 % (w/w), Al2O3 4.05 % (w/w), SO3 (sulphate) 3.47 % (w/w), MgO 2.25 % (w/w), Fe2O3 1.81 % (w/w), CaO (free) 1.40 % (w/w), CO2 1.29 % (w/w), Na2O 0.60 % (w/w), TiO2 0.34 % (w/w).
- Impurities:
As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn sum < 0.1%
- Lot/batch No.: BMA/F/2009
- Expiration date of the lot/batch: 15 years / 12/2024
- Stability under test conditions: stable
- Appearance: black powder
- Storage condition of test material: in PE container at room temperature

Test animals

Species:
rat
Strain:
other: Wistar-Han (outbred, SPF quality - guaranteed)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Lysolaje 15, 165 00 Praha 6, závod Koleč u Kladna Czech Republic, RČH CZ 21760152
- Age at study initiation: 6-7 weeks
- Fasting period before study: no
- Weight at study initiation: At the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20% of the mean weight.


- Housing: Animals were housed in plastic cages (40x25x20cm) containing sterilised clean shavings of soft wood - 2 rats of the same sex in one cage.
- Diet (e.g. ad libitum): Complete pelleted diet for rats in SPF breeding - ST 1 BERGMAN, manufacturer:
Ing. Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using.

- Water (e.g. ad libitum): Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law. Water was sterilised before using.
- Acclimation period: at least 5 days
- Identification:
Identification of animals was made by colour marks on fur (system 1 – 10), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance and mark of group.


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

IN-LIFE DATES:
Study Time Schedule
Test substance delivery: 6.1.2010
Date of animal arrival: 2.6.2010
Start of administration: 9.6.2010
End of administration: 9.9.2010
Cllinical examination:
main groups 2.6. – 10.9.2010
satell. groups 2.6. – 6.10.2010
Urinalysis:
main groups 6.9. – 9.9.2010
satell. groups 4.10. – 5.10.2010
Blood taking and necropsies:
main groups 7.9. – 10.9.2010
satell. groups 5.10. – 6.10.2010
Histopathological examination: 10.10. – 5.12.2010
Evaluation of results and final report elaboration: 20.10. – 6.1.2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1mL per 100 g of body weight.
The application form of the test substance (suspension in olive oil) was prepared daily just before administration. The mixture was mixed for 10 minutes (cca 1000 rpm). The vehicle control group was administered by olive oil in the same volume.

VEHICLE
- Olive oil (Olivae oleum raffinatum) - Batch No.: 4726901, 4874601
Manufacturer: Dr. Kulich Pharma, s.r.o., Hradec Králové, Czech Republic
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test substance consists of various oxides insoluble in the application form (olive oil). A suitable analytical method cannot be found for homogenity and stability testing. Since undissolved particles of the test substance are easily visible in the application form,
homogeneity was checked by eye (suspension were mixed for 10 minutes by magnetic stirrer – 1000 rpm). Stability of the test substance in the application form cannot be verified but there is no indication that a mixture of rigid oxides would be unstable in its solution (in olive oil) for that short time period (2 hours).
Duration of treatment / exposure:
90 days
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 – 10.00 am)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
160 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
400 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 females and 10 males per group; 6 females and 6 males in satellite group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses for the 90-day study were chosen with respect to the results of study phase Dose-range finding experiment.
The dose-range finding experiment with 28-day application period was performed with 4 groups of treated animals without control group.
The doses for the dose-range finding experiment were chosen with respect to the preliminary results of acute toxicity study.
Appropriate dose levels of the test substance for the 28-day dose-range finding experiment were chosen as follows: 125, 250, 500 and 1000 mg/kg/day
- Post-exposure recovery period in satellite groups: 28 days



Examinations

Observations and examinations performed and frequency:
HEALTH CONDITION CONTROL: Yes
- Time schedule: daily during the check-in, acclimatisation period, during the administration and during the recovery period in groups

CLINICAL OBSERVATION: Yes
- Time schedule: daily during the administration period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first application and then weekly

MORTALITY: Yes
- Time schedule: daily during the acclimatisation and recovery periods and twice daily during the treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and immediately before euthanasia

FOOD CONSUMPTION:
- Food consumption for animal/day was calculated from average values of each group.
- Time schedule: weekly

FOOD EFFICIENCY:
- Calculation of food conversion in %: weight increment/food consumption x 100.
- Time schedule: weekly

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study
- Anaesthetic used for blood collection: Yes, light ether narcosis
- Animals fasted: Yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study
- Animals fasted: Yes
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: 90th (main group); 118th (satellite group) day of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

FUNCTIONAL OBSERVATION: Yes
- Time schedule: at the last week of administration period and recovery period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the 1st week of of application and at the end of administration period and recovery period (at the control and the highest dose level).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Biometry of Organs - the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart 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.

Pathological Examination - during the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out.

HISTOPATHOLOGY: Yes. Organs for histopathological examination - see table No.5

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
The oral administration of the test substance at the dose levels 160, 400 and 1000 mg/kg/day did not cause mortality and produced no adverse significant changes detected in body weight, food consumption, food conversion, water consumption, health condition control, clinical observation, functional observation, ophtalmological examination and macroscopic structure of organs.

Clinical observations did not demonstrate responses of biological importance. Occurence of vocalisation during handling of treated animals was only sporadic in females.

BODY WEIGHT AND WEIGHT GAIN, FOOD CONSUMPTION
Effect of the test substance administration on growth of animals was rather positive and it was more pronounced in males. In spite of slightly decreased food intake of treated males their body weights were slightly higher or ballanced compared to the control males for the whole application and observation periods. In treated females body weights were analogous or slightly lower in comparison with control even if their food consumption was slightly decreased.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects were detected.

HAEMATOLOGY
Haematological examination showed reversible effect on red blood component only in females – haemoglobin concentration was significantly depressed at the middle and the highest dose level. It was not accompanied by abnormal values of other parameters of red blood component. White blood component was significantly changed in males and females only at the end of observation period: total leucocyte count elevated in females was accompanied by changes of differential leucocyte count (decrease of lymphocyte portion), whilst in males abnormal differential count (increase of lymphocyte portion and decrease of granulocyte portion) without change of total white cell count was recorded. Microscopical examination of organs did not revealed increased occurence of inflammation which could cohered with this shift in differential leucocyte count. Haemocoagulation was significantly influenced in the following way: platelet count was decreased in both sexes at the end of observation period, fibrinogen was decreased in males of the lowest dose level and in treated females at the end of observation period and protrombin time was changed (shortening) only in females of the highest dose level. None of above mentioned changes of haematological parameters occured in a dose related manner and had mostly delayed character.


CLINICAL CHEMISTRY
Biochemical examination of blood revealed statistically significant changes in groups of the middle and the highest dose level. Some of them probably related to chemical composition of the test substance: serum concentration of calcium ions was reversibly increased in both sexes and in males it occurred in a dose related manner, elevation of serum phosphorus ions was detected at the end of observation period. Calcium and phosphoric oxides belong to main components of the test substance. Also other electrolyte disturbance diagnosed in males of the highest dose level – hyponatraemia had connection with chemical composition of the test substance: higher intake of potassium oxide (another component of the test substance) probably caused reduction of serum sodium ions concentration. Reduction of sodium level had irreversible character – concentration was slightly decreased at the end of administration period but significantly decreased at the end of observation period. Sodium homeostasis is vital to the normal physiologic function of cells so diffenrences in this parameter are important and can be considered to be of toxicological significance. Irreversible increase of creatinine concentration in females can bear evidence of test substance toxic effect on kidneys because according to the literary data creatinine level has a great positive predictive value of kidney changes.

URINALYSIS
During urinalysis only significant decrease of urine volume was detected in males at the end of observation period. It could be excessive reparative change because at the end of administration period the urine volume in males of the highest dose level was slightly increased in males of the highest dose level.

ORGAN WEIGHTS
Organ weight differences between the control and treated males were recognisable in biometry of organs at the end of observation period: absolute weight of treated males heart, testes and epididymides were significantly increased compared to control. In females with lower body weight (at the highest dose level) significant decrease of absolute weight of spleen and kidneys was recorded. Significant relative enlargement was found out in liver of males at the end of administration period and in heart of females at the end observation period. Adrenal glands of the highest dose level males were relatively reduced at the end of administration period. It cohered with higher body weight – weight of adrenal glands does not change generally with change of body weight. At the end of treatment period the weights of testes, epididymides and prostate gland were insignificantly reduced with compensation or even elevation at the end of observation period. In the absence of any supporting histopathology, the weight changes of most above mentioned organs were not considered to be toxicologically relevant. Pathological examination revealed effect of the test substance on microscopical structure of liver: slightly increased occurence of focal fatty changes (presence of spherical fatty vacuoles in cytoplasm of hepatocytes - situated irregularly or in peripheral part of lobuli) in both sexes of the highest dose level. This microscopical lesion was recorded in the treated and also in the control animals during examination of specially stained cryotome sections (by oil red) but with various incidence.

HISTOPATHOLOGY
Histological examination of kidneys did not revealed morphological affections. Reversible decrease of urea and cholesterol concentration accompanied by increased concentration of bilirubin (in bilirubin - dose related) was recorded in males of the highest dose level. These changes could relate to liver metabolism changes.

Effect levels

Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test substance administration had not negative effect on body weight, food consumption, food conversion, water consumption, mortality, health condition, clinical examination, parameters of functional and ophtalmological examination and macroscopical structure of organs of animals.
The test substance, Ashes (residues) – Biomass Combustion, after 90-day oral application caused significant changes of haematological parameters (changes of fibrinogen, haemoglobin concentration platelet count and differential leucocyte count), biochemical parameters (hyponatraemia, increased creatinine, changes of urea, cholesterol, bilirubin, calcium, phosphorus and chloride ions concentration) and biometry of organs (changes of weights of esp. adrenal glands, liver and heart).
The test substance had influence on microscopical structure of liver (focal fatty changes).

The highest incidence of statistically significant differences was recorded at the highest dose level (sometimes with delayed character) while most of changes which were found out at the middle and the lowest dose level had only mild intensity without adverse alteration of animal organism.

The value of NOAEL (No Observed Adverse Effect Level) for MALES and FEMALES was established as 400 mg/kg/day.
Executive summary:

Introduction

   The test substance, Ashes (residues) - Biomass Combustion, was tested for subchronic toxicity using the Method B.26 Sub-Chronic Oral Toxicity Test: Repeated Dose 90-day Oral Toxicity Study in Rodents, Council Regulation (EC) No. 440/2008, Published in O.J. L142, 2008. This sub-chronic oral toxicity test method is a replicate of the OECD Test Guideline No. 408: Repeated Dose 90-day Oral Toxicity Study in Rodents, Adopted 21st September 1998.

Methods

   Wistar rats of SPF quality were used for testing. The test substance was administered in olive oil by stomach tube; oral application of rats was made daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 10 males and 10 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 160, 400, 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 administration period lasted 90 days. After that animals of main groups were sacrificed and satellite animals were observed for the next 28 days without treatment.

   

   The dose levels for the main study - 160, 400, 1000 mg/kg/day were chosen on the basis of results of dose-range finding experiment (see the Annex 1).

   During the 90-day study clinical observation and health status control were performed daily. The body weight, food consumption were measured weekly and the detailed clinical observation was carried out in the same time interval. Water consumption was measured twice a week. Ophthalmologic examination was done at the beginning and at the end of the study. Before the end of study the functional observation was accomplished. The study was finished by urinalysis, haematological and biochemical analysis, and gross necropsy of animals. The selected organs for weighing and histopathological examination were removed.

Results

    The oral administration of Ashes (residues) - Biomass Combustion to rats by gavage for period of 90 consecutive days at the dose levels 160, 400, 1000 mg/kg/day did not cause any mortality. No negative treatment-related effects were detected during body weight, food consumption, food conversion and water consumption control, health condition control, functional observation, ophtalmological examination and macroscopical examination of organs and tissues.        

    Clinical status of animals after application, biochemical blood parameters, urinalysis parameters, biometry and microscopical structure of organs were not seriously affected by treatment of the test substance at the dose level 160 mg/kg/day. Some haematological parameters (decrease of fibrinogen in males and haemoglobin concentration in females) in animals at the dose level 160 mg/kg/day were influenced by administration of the test substance.       

    Clinical status of animals after application, urinalysis parameters and microscopical structure of organs were not seriously affected by treatment of the test substance at the dose level 400 mg/kg/day. Haematological parameters (decreased haemoglobin in females), blood biochemical parameters (decreased serum urea concentration in males, decreased serum chloride ions concentration in females) and biometry of adrenal glands (decreased absolute weight in males) in animals at the dose level 400 mg/kg/day were influenced by administration of the test substance. 

   Clinical status of animals after application were not seriously affected by treatment of the test substance at the dose level 1000 mg/kg/day. Haematological parameters (reversible decrease of haemoglobin concentration in females, decrease of protrombin time value in females; delayed increase of platelet count in males and females, decrease of percentual portion of granulocytes and increase of lymphocytes in males, increase of total leucocyte count in females, decrease of percentual portion of lymphocytes in females and increase of fibrinogen value in females) and blood biochemical parameters (irreversible decrease of sodium ions concentration in males and increase of serum creatinine concentration in females; reversible decrease of serum cholesterol concentration in males, decrease of serum urea concentration in males, increase of serum bilirubin concentration in males and increase of serum calcium ions concentration in males and females; delayed decrease of serum ALP activity in females and increase of serum phosphorus ions concentration in males and females), urinalysis parameters (delayed decrease of urine volume in males), biometry of organs (reversible decrease of absolute and relative weight of adrenal glands and increase of relative weight of liver in males, decrease of absolute weight of spleen and kidneys in females; delayed increase of absolute weight of heart, testes and epididymides in males and increase of absolute and relative weight of heart in females) and microscopical structure of liver (slightly increased occurrence of microscopical focal fatty changes in liver in males and females) in animals at the dose level 1000 mg/kg/day were influenced by administration of the test substance.