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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.8.2009-23.3.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance with internationally valid GLP principles.

Data source

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

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:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Fluidized Bed Combustion (FBC) Fly Ash
- Substance type: technical product
- Physical state: solid
- Appearance: Light grey powder
- Chemical structure: Complex product of oxides
- Main components: SiO2 - 35,59%, Fe2O3 - 7,69%, CaO (total) - 19,46%, Na2O - 0,53%, P2O5 - 0,4%, CO2 - 1,11%, CaO (free) - 8,41%, Al2O3 - 20,88%, TiO2 - 4,93%, MgO - 0,41%, K2O - 0,43%, SO3 (sulphate) - 6,89%
- Impurities: Metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn - sum < 0,1%
- Lot/batch No.: FBC/230309/T1
- Expiration date of the lot/batch: 03/2024
- Stability under test conditions: stable
- Storage: The substance was in PE container at room temperature.

Test animals

Species:
rat
Strain:
Wistar
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: none
- Housing: Animals were housed in plastic cages 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
- Water (e.g. ad libitum): Drinking water
- Acclimation period: at least 5 days

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

STUDY TIME SCHEDULE
Date of animal arrival: 24. 8. 2009
Acclimatisation: 24. 8. – 8. 9. 2009
Start of administration: 9. 9. 2009
End of administration: 10. 12. 2009
Clinical observation: main groups 9. 9. – 10. 12. 2009, satell.groups 9. 9. – 10. 12. 2009
Urinalysis: main groups 7. – 10. 12. 2009, satell.groups 4. – 5. 1. 2010
Blood taking and necropsies: main groups 8. – 11. 12. 2009, satell.groups 5. – 6. 1. 2010

Administration / exposure

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

VEHICLE
- Olive oil (Olivae oleum raffinatum) - Batch No.: 4683401
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days per week at the same time
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
800 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 females and 10 males per group; 5 females and 5 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 derived from the limit dose 1000 mg/kg/day (according to the guideline).
- 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

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

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: control and the highest dose level

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

MORTALITY: Yes
- Time schedule: daily

FUNCTIONAL OBSERVATION: Yes
- Time schedule: at the end of administration period and recovery period
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.
Pathological Examination - the 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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHT AND WEIGHT GAIN
- Effect of the test substance administration on growth of animals was rather positive. In spite of decreased food intake of treated males their body weight gains were higher than in control males for the whole of the application period. In survival period body weight increments of the treated and the control males were analogous. Body weights of treated and control females were ballanced during all the study.

CLINICAL OBSERVATION
- Clinical observations revealed only slight influence of the test substance on clinical status of treated animals. Occurence of chromodacryorrhea in treated males was slightly increased (esp. in males at the highest dose level). In treated females this clinical change occured only sporadically. Chromodacryorrhea - excessive outpouring of red secretion of the Harderian gland could be provoked by non-specific stimuli such a stress. After the end of application of the test substance this clinical symptom disappeared - effect had reversible character.

HAEMATOLOGY
- Haematological examination showed an effect on red blood component, white blood component and haemocoagulation. None of observed changes was dependent on dose level. Reversible effect on red blood component was evident in males of all dose levels: statistically significant increase of haematocrit or haemoglobin value without change of total red cell count. In females this symptom had delayed character: increased value of haemoglobin exceeded the historic kontrol limits. Polyglobulia (increased amount of hemoglobin) was probably only relative. It could be caused by decreased volume of plasma which could also cohered with hyperalbuminaemia. Mean erythrocyte volume was significantly changed in males at the end of observation period and in females of the middle dose level at the end of application period. White blood component was significantly changed in females: at the end of application period decrease of total leucocyte count at the highest dose level was observed (insignificant decrease was observed also at the lowest and the middle dose level) whilst increase of total leucocyte count occured at the end of observation period. This increased value was out of historic control and it was accompanied by significant increase of monocytes portion. Microscopical examination of organs did not revealed increased occurence of inflammation which could cohered with this shift in differential leucocyte count. Also platelet count was changed in females: insignificant decreasing at the end of application period and marked (out of historic control) increasing at the end of observation period.

CLINICAL CHEMISTRY
- Statistically significant changes without dose dependence were registered during biochemical examination of blood: the value of albumin was reversibly increased in males of the lowest and the middle dose level and in females of the middle dose level. In females this difference was accompanied by dose dependent increased level of total protein. Hyperalbuminaemia was not considered to be biologically significant, it was probably related to haemoconcentration analogous to increased hemoglobin. In females hyponatremia was recorded and it had delayed character. Values of none biochemical parameters exceeded the reference interval.

URINALYSIS
- Dose dependent partly irreversible decrease of urine pH was detected during urinalysis of females. This change was statistically significant at the middle and the highest dose level. Decreased value of urine pH could be related to chemical composition of the test substance. In males urine pH of treated and control males was similar. Volume of urine was significantly increased in males at the end of observation period. Occurence of leucocytes in urine was increased especially in males of the middle and the highest dose level but this change was not accompanied by any histopathological findings in kidneys.

ORGAN WEIGHTS
- Absolute weight of treated males organs were increased compared to control whilst relative weights of treated males organs were lower than in control. In females only one significant difference - increase of absolute weight of brain was recorded only at the lowest dose level

HISTOPATHOLOGY: NON-NEOPLASTIC
- Presence of microscopical lesions in liver - focal fatty changes (presence of spherical fatty vacuoles in cytoplasm of hepatocytes - situated irregularly or in peripheral part of lobuli) were recorded in treated and also in control animals. Casual histological examination of Haematoxylin - Eosin stained sections revealed focal fatty changes only in males of the highest dose level. During examination of specially stained cryotome sections (by oil red) cytoplasmic vacuoles filled with fat were found out in all examined animals with various incidence. In control males and in females of the lowest and the middle dose level only sporadic hepatocytes were affected. In males of the lowest and the middle dose level and females of the highest dose level occurence of affected hepatocytes was slightly increased. Incidence of fat foci in males of the highest dose level was significantly increased compared to control.

HISTORICAL CONTROL DATA:
- Source of data: haematological values of control animals from five 90-day oral toxicity studies and values of biochemical examination of control animals from five 90-day oral toxicity studies. Calculation of interval: mean of group ± 2×standard deviation (10th – 90th percentile).

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxic effect observed.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test substance administration had not effect on food consumption, food conversion, mortality, health condition, parameters of functional and ophtalmological examination of animals. Body weight and accompanied biometry of organs was not negatively influenced by the test substance treatment.
The test substance, Fluidized Bed Combustion (FBC) Fly Ash, after 90-day oral application caused significant changes of haematological (haematocrit, haemoglobin, MCV, total leucocyte count, monocytes portion) and biochemical parameters (albumin, sodium) at all dose levels. But exceeding of reference interval was recorded only in females at the end of observation period (MCV, haemoglobin, total leucocyte count and platelet count).
The urinalysis demonstrated effect of the test substance on properties of urine (pH, presence of leucocytes).
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 200 mg/kg/day.

Executive summary:

Introduction

The test substance, Fluidized Bed Combustion (FBC) Fly Ash, 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 5 males and 5 females. Main groups contained 3 treated groups (doses 50, 200, 800 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 (800 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 - 50, 200, 800 mg/kg/day were chosen on the basis of results of Fluidized Bed Combustion (FBC) Fly Ash - Repeated Dose 90-day Oral Toxicity Study – Dose–range finding experiment .

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 Fluidized Bed Combustion (FBC) Fly Ash to rats by gavage for period of 90 consecutive days at the dose levels 50, 200, 800 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 biometry of organs.        

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 50 mg/kg/day.          

Clinical status of animals after application were unaffected by treatment of the test substance at the dose level 200 mg/kg/day.

Haematological parameters (increase of haematocrit in males) and blood biochemical parameters (increase of albumin in males), some urinalysis parameters (slight decrease of pH in females), macroscopical structure of liver (change of colour in males) and microscopical structure of liver (fatty change - sporadically in males) in animals at the dose level 50 mg/kg/day were influenced by administration of the test substance.

Haematological parameters (increased haemoglobin in males, decreased of MCV in females) and blood biochemical parameters (increased albumin in both sexes), urinalysis parameters (decrease of pH in females, presence of leucocytes in males), macroscopical structure of stomach (congestion or susp. mucosal haemorrhages - slightly in males) and liver (change of colour and marked structure in both sexes) and microscopical structure of liver (fatty change - slightly in males) in animals at the dose level 200 mg/kg/day were influenced by administration of the test substance. 

Clinical status of animals after application (slightly in males and sporadically in females),  haematological parameters (HCT, MCV in males, haemoglobin, total and differential leucocyte count, platelet count in females) and blood biochemical parameters (total protein in both sexes, sodium ions in females), urinalysis parameters (decrease of pH in females, increase of volume in males, presence of leucocytes in both sexes), biometry of organs (decrease of relative weight of kidney), macroscopical structure of stomach (congestion or susp. mucosal haemorrhages - slightly in males) and liver (change of colour and marked structure in males and females) and microscopical structure of liver (fatty change, esp. in males) in animals at the dose level 800 mg/kg/day were influenced by administration of the test substance.    

Conclusion

The test substance administration had not effect on food consumption, food conversion, water consumption, mortality, health condition, parameters of functional and ophtalmological examination of animals. Body weight and accompanied biometry of organs was not negatively influenced by the test substance treatment.

The test substance, Fluidized Bed Combustion (FBC) Fly Ash, after 90-day oral application caused significant changes of haematological (haematocrit, haemoglobin, MCV, total leucocyte count, monocytes portion) and biochemical parameters (albumin, sodium) at all dose levels. But exceeding of reference interval was recorded only in females at the end of observation period (MCV, haemoglobin, total leucocyte count and platelet count).

The urinalysis demonstrated effect of the test substance on properties of urine (pH, presence of leucocytes).

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 200 mg/kg/day.