Ashes (residues), nonhazardous municipal solid waste

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:

8.1.2008-2.4.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues)

EC Number:

268-627-4

EC Name:

Ashes (residues)

Cas Number:

68131-74-8

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): Ashes (residues)
- Substance type: technical product
- Physical state: solid
- Main components: SiO2 (42.12%), Al2O3 (32.16%), Fe2O3 (10.88%), TiO2, MnO, MgO, CaO, Na2O, K2O, SO3 - constituents with content less than 10%
- Expiration date of the lot/batch: unlimited
- Appearance: greyish black powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., 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 SPF animal room, 2-3 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood.
- Diet - ad libitum, complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN), producer: Mlýn Kocanda, Jesenice by Prague was used. Diet was sterilised before using. (Capacity of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%;
Composition of food: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex)
- Water - ad libitum, free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Water was sterilised before using.
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3oC
- 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
Test substance delivery: 16. 10. 2007
Dose-range finding experiment: 9. 1. – 24. 1. 2008
Main study:
Date of animal arrival: 13. 2. 2008
Start of administration: 19. 2. 2008
End of administration: 20. 3. 2008
Clinical observation: 19. 2. - 21. 3. 2008 main groups, 19. 2. - 2. 4. 2008 satellite groups
Urinalysis: 17. - 20. 3. 2008 main groups, 31. 3. – 1. 4. 2008 satellite groups
Blood taking and necropsies: 18. – 21. 3. 2008 main groups, 1. – 2. 4. 2008 satellite groups
Histopathological examination: 13. 5. – 16. 7. 2007

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose (in water for injection)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Concentrations of solutions at every dose levels were adjusted to ensure the administration equals 1 mL per 100 g of body weight. The application form (test substance suspension in 0.5% methyl cellulose) was prepared daily just before administration. The vehicle control group was administered by 0.5% methyl cellulose in the same volume. The application form of the test substance was prepared daily before administration; these suspensions were mixed for 10 minutes by magnetic stirrer.


VEHICLE
The test substance was administered suspended in 0.5% methyl cellulose (in water for injection).

Water for injectione - Aqua pro injectione
Manufacturer: Ardeapharma a.s., Ševětín, Batch No.: 0102101007

Methylcellulosum
Manufacturer: Dr. Kulich Pharma, s.r.o., Hradec králové, Batch No.: DT157078

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The treated and control groups were administered daily for the period of 28 days.

Frequency of treatment:

The animals were treated 7 days per week at the specified time (8.00 – 10.00 am).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
250 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 male and 5 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the dose-range finding the following the dose levels - 250, 500 a 1000 mg/kg/day were chosen for the main 28-day Oral Toxicity Study.
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 14 days after the end of application

Positive control:

none

Examinations

Observations and examinations performed and frequency:

GENERAL CLINICAL OBSERVATION: Yes
- Time schedule: All rats were observed daily during the administration period.
- This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. This was made at specified time after application every day – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: This observation was carried out before the first application and then weekly.
- At the first part of observation behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour.
- The second part was the observation during removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- The body weight of animals was recorded on automatic balances with group average computing module. All animals were weighed immediately before euthanasia too. Weight increment was computed as an average per group per day (in grams).


FOOD CONSUMPTION:
- Time schedule: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption for animal/day was calculated of average values of each group.
- In a specified day every week the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed. Average values were calculated for each week of the study.


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

WATER CONSUMPTION:
- Time schedule for examinations: twice a week
- Drinking water consumption was recorded. Average values (water consumption per animal and per day) were calculated for each week of the study.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: : 29th day of study (main groups) or 43rd day of study (satellite groups)
- 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: 29th day of study (main groups) or 43rd day of study (satellite groups)
- Animals fasted: Yes
- How many animals: all animals


URINALYSIS: Yes
- Time schedule for collection of urine: 28th day of study (main groups) or 42rd day of study (satellite groups)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

MORTALITY CONTROL: Yes
- Time schedule: daily

HEALTH CONDITION CONTROL: Yes
- Time schedule: daily - during the acclimatization and the experimental part
- The health condition was controlled daily during the check-in, acclimatisation period, during the administration and during the recovery period in groups. Pre-experimental observation of all rats was performed to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. In administration period this observation was performed before application.

FUNCTIONAL OBSERVATION: Yes
- Time schedule: in the last week of administration period (main groups) or in last week recovery period (satellite groups)
- During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) pectoral legs, 2) pelvis legs, 3) all four legs. Grip power was expressed in Newtons.
-

Sacrifice and pathology:

At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of 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, 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.

GROSS PATHOLOGY: Yes
- 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. Organs for consequent histopathological examination were taken out and stored in containers with fixative (neutral 4% formaldehyde).

HISTOPATHOLOGY: Yes (see table No.5)
- Tissue specimens fixed in 4% neutral formaldehyde were processed by routine paraffin technique and stained by hematoxyline-eosine. Cryotome sections of liver and kidneys were stained by oil red for neutral lipids.

Statistics:

The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with satellite treated group.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of medians or averages.

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:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

DISCUSSION OF THE RESULTS

The toxic effect of the test substance was not expressed at any dose level.
There were no unscheduled deaths during the test. No clinically observed signs of toxicity were detected. The animal’s heath condition was very good during the whole study.
Functional observation evidenced no effect of the test substance. All inter and intra group differences in behavioural scores (upstanding, emiction, defecation) and sensory reactivity were considered to be a result of normal variation for rats of the strain and age used and were no toxicological importance. There were no treatment-related changes in functional performance parameters measured (grip strength).
No adverse effect on body weight development was detected. The body weight of all test groups was well-balanced without statistically significant difference during the whole application and recovery period. The intergroup difference was minimal and, in isolation, was regarded as fortuitous. No significant differences of average body weight increment were recorded in treated groups of males and females (in comparison with control).
No adverse effect on dietary intake or food conversion was detected. Food consumption of all groups was well-balanced. Sporadic slight intergroup reduction or increasing of food consumption and food conversion males were considered to be a result of normal variation, which could not be related with administration of the test substance.
Daily inspection of water consumption revealed no significant intergroup differences during the application period. Slight decrease of consumption was recorded in males at the highest dose level during the application period. Urinalysis showed statistically significantly decreased volume urine in males (without changed pH or specific weight) and sporadic presence of urobilinogen, blood and leucocytes in urine at the highest dose level. This changed parameter together with decreased water consumption and macroscopic findings (changed colour) could be attributed to treatment-related effect on urine excretion system. Expected microscopic changes at the highest dose level were found out only sporadically. Urinalysis detected no treatment-related other effects in males and no effect in females.
The haemocoagulation examination detected statistically significant changes only in males. Prolongation of protrombine time and increased fibrinogene score were recorded at the middle and the highest dose level at the end of application period. Prolongation prothrombine time was also recorded in satellite treated males after 14-day recovery period – the effect of the test substance on this blood component was protracted. All changed parameters were inside physiologic limit. These changes of blood coagulation were not approved in clinical biochemistry. Activity of hepatic enzymes was similar compared with males in the control group. Increased platelet count was recorded at the lowest dose level but related parameters were unchanged.
The haematology parameters of red blood cells were statistically significant changed only in satellite males. Decreased value of total erythrocyte count and accompanying increase of mean corpuscular volume were measured in treated males at the end of recovery period, but expected changes of haematocrite and haemoglobine volume was not detected.
Statistical analysis of the other blood chemical parameters revealed significant intergroup differences in satellite females at the end of recovery period. Glucose concentration and activity of ALP were increased. Changes of differential leucocyte count with statistical significance were also recorded. All these inter group differences were considered to be adaptative response to stress without toxicological significance.

Biometry of organs detected significant inter group differences in the weight of endocrines glands. Absolute or relative weight of pituitary glands was decreased in animals of both sexes at the end of the application period. Statistically significant decrease of relative weight was recorded in males at the middle dose and in females at the lowest and the highest dose levels. Slight increase of presence cyst and pseudocysts in pituitary gland (histopathological findings) was recorded at all treated groups and control groups. These morphological changes are commonly observed in employed strain of rats. Also differences in incidence between control and treatment groups are considered to be no toxicological significance. Decrease of absolute and relative weight of adrenal glands was recorded in females at the end of the recovery period, but without related macroscopic abnormalities or microscopic changes.

Histopathological examination revealed inflammation of liver in treated and control animals of both sexes. Slight dose dependence was found out at the end of the application period. Slightly increased frequency of focal inflammation persisted in satellite males. Due to absence of biochemical parameters showing functional disturbance, this was considered to be no toxicological importance.
Dose dependence increased presence of oedema in prostate gland (presence of eosinophile liquid in intercellular tissue) was recorded in males. This histopathological change can be the first stage of inflammation. Inflammation in prostate gland was more frequent change in males and hydrometra of uterus in females. Frequencies of these microscopic findings in genital tract were similar in treated and control groups.
Other changes were recorded only sporadically: increased absolute weight of thymus in females without other related findings, sporadic histopathological findings in heart, in small intestine, stomach or thymus. This low incidence of abnormality without dose-related response was considered to be no toxicological importance.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: No toxic effect observed.

2

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

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 oral administration of the test substance Ashes (residues) to rats by gavage for a period of twenty-eight consecutive days at dose level 250, 500 and 1000 mg/kg/day produced no toxicologically significant changes in the parameters measured. No major functional changes in any organ systems or severe organ dysfunction were detected. Consistent changes in clinical biochemistry, haematology and urinalysis parameters which indicate organ dysfunction were not recorded in any dose level. Histopathological examination induced slight pathological changes in males (oedema in prostate gland), which could be related with administration of the test substance.

Based on the results of laboratory investigations in clinical biochemistry, haematology and urinalysis and histopathological examination the following conclusion about NOAEL can be sugested:

The NOAEL (No Observed Adverse Effect Level) for MALES is 500 mg/kg/day and for FEMALES is higher or equal to 1000 mg/kg/day.

Executive summary:

Introduction

The test substance, Ashes (residues), was tested for subacute toxicity using the method B.7.Repeated Dose (28-days) Toxicity (Oral), Directive 96/54/EC.

 

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered as suspension in 0.5% methyl cellulose using a 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 5 males and 5 females; each satellite group consisted of 5 males and 5 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 administration period lasted 28 days. After that animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.

 

Doses for the main study - 250, 500, 1000 mg/kg/day were chosen on the basis of results of dose-range finding experiment.

 

During the 28-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. 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 histopathology examination were removed.

 

 

Results

There were no unscheduled deaths during the test. No clinically observed signs of toxicity were detected. The animal’shealthcondition was very good during whole study and functional observation evidenced no effect of the test substance.

No adverse effect on body weight development was detected. The body weight and body weight increment of all test groups was relatively well-balanced during whole application and recovery period. No adverse effect on dietary intake or food conversion was detected.

 

The haemocoagulation examination detected significant changes - prolongation of protrombine time and increased fibrinogene score were recorded in males of the middle and the highest dose level at the endof application period. Prolongation of protrombine time was also measured in males after 14-day recovery period – the effect of the test substance on this blood component was protracted. No other accompanyingchanged parameters were found.

 

The males treated by the test substance at the highest dose level showed changed parameters in urinalysis, which could indicate slight effect on urine excretion system – decrease of water consumption, decreased volume of urine.

   

Biometry of organs detected significant inter group differences in the weight of some endocrine glands. The microscopic changes were detected only in pituitary gland. Slight increase of presence cyst and pseudocysts was recorded at all treated groups and control groups. These morphological changes are commonly observed in employed strain of rats. Also differences in incidence between control and treatment groups are considered to be of no toxicological significance.

Histopathological examination revealed inflammation of liver both in treated and control animals of both sexes (with protracted effect in males). Due to absence of other changed biochemical parameters showing functional disturbance, this was considered to be of no toxicological importance. Dose dependence increased presence of oedema in prostate gland was recorded in males. This histopathological change can be the first stage of inflammation.

 

Overall assessment

The oral administration of the test substance Ashes (residues) to rats by gavage for a period of twenty-eight consecutive days at dose level 250, 500 and 1000 mg/kg/day produced no toxicologically significant changes in the parameters measured. Nomajor functional changes in any organ systems or severe organ dysfunction were detected. Consistent changes in clinical biochemistry, haematology and urinalysisparameters, which would indicateorgandysfunction were not recorded in any dose level. Histopathological examination induced slight pathological changes in males (oedema of prostate gland), which could be related with administration of the test substance.

 

Based on the results of histopathology examination and laboratory investigations in clinical biochemistry, haematology and urinalysis it could be done the following conclusion about NOAEL:

  

The NOAEL (No Observed Adverse Effect Level) for MALES is 500 mg/kg/day and for FEMALES is higher or equal to 1000 mg/kg/day.