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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12.1.2010-1.8.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

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

Constituent 1
Reference substance name:
Slimes and Sludges, blast furnace and steelmaking
EC Number:
266-006-2
EC Name:
Slimes and Sludges, blast furnace and steelmaking
Cas Number:
65996-73-8
Molecular formula:
not applicable
IUPAC Name:
Slimes and sludges, blast furnace and steelmaking
Constituent 2
Reference substance name:
266-066-2
IUPAC Name:
266-066-2
Details on test material:
- Physical state: solid
- Composition of test material, percentage of components: Fe (total) 54.29% (mainly as oxides), CaO 10.17%, Zn 5.25%, MgO 4.32%, C 2.06%, SiO2 1.64%, Mn 0.69%, K2O 0.126%, Na2O 0.239%, Al2O3 0.16%
- Lot/batch No.: 21.10.2009
- Expiration date of the lot/batch: unlimited
- Stability under test conditions: unlimited
- Storage condition of test material: stored in PE container at room temperature

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: VELAZ s.r.o., Kolec u Kladna, Czech Republic (SPF breeding)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: cca 206-246 g M and cca 150-176 g F
- Housing: Animals were housed in SPF (Specified Pathogen Free) animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood.
- Diet (e.g. ad libitum): Complete pelleted diet for rats in SPF breeding - ST 1 BERGMAN ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: suspension was mixed for 10 minutes by magnetic stirrer

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food): olive oil

VEHICLE
- Concentration in vehicle: Concentrations of solutions at every dose levels were adjusted to ensure the administration equals 1 mL per 100 g of body weight.
- Amount of vehicle (if gavage): Amount of vehicle at every dose levels were adjusted to ensure the administration equals 1 mL per 100 g of body weight.
- Lot/batch no. (if required): 46 83 401
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 d
Frequency of treatment:
1×daily 7 times per week at the specified time (8:00-10:00 am)
Doses / concentrationsopen allclose all
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: on the basis of range-finding test results
- Post-exposure recovery period in satellite groups: 14 days after end of treatment
Positive control:
no

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily - during the administration period

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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.

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.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 29th day of study (main groups) and 43rd day of study (satellite groups)
- Animals fasted: Yes (18 hours but drinking water ad libitum)
- Anaesthetic used for blood collection: Yes (light ether narcosis)
- Parameters RBC (total erythrocyte count), MCV (mean corpuscular volume), haematocrit, haemoglobin, WBC (total leucocyte count), platelet count, APTT (activated partial thromboplastin time), prothrombin time, fibrinogen, granuklocytes, lymphocytes and monocytes were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 29th day of study (main groups) and 43rd day of study (satellite groups)
- Animals fasted: Yes (18 hours but drinking water ad libitum)
- Parameters glucose, total cholesterol, urea, total bilirubin, aspartate aminotransferase, alanine amonitransferase, alkaline phosphatase, calcium, phosphorus, total protein, protein-abumin, creatinine, sodium, potassium and chlorine were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 28th day of study (main groups) and 42nd day of study (satellite groups)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters volume, colour, cloud, odour, glucose, protein, bilirubin, urobilinogen, pH, specific gravity, blood, ketones, nitrites and leucocytes were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of administration period at main groups or at the end of recovery period at satellite groups
- Dose groups that were examined: all dose groups including control groups
- Battery of functions tested: approximation, contact point, reaction to noise and pain, pupillary reflex, upstading, emiction, defaecation, grip strenth (forelegs, hind-legs and total)

MORTALITY:
- Time schedule for examinations: daily during the treatment and recovery period

HEALTH CONDITION CONTROL:
- Time schedule for examinations: daily - during the acclimatization and the experimental part
Sacrifice and pathology:
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. 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. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde).

HISTOPATHOLOGY: Yes
Tissue specimens fixed in 4% buffered 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.

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):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was slightly unbalanced in female.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematology examination detected significant irreversible effect on red blood cells parameters in both sexes of all treated groups but markedly at the highest dose level. The increased value of hematocrit and haemoglobin were recorded in both sexes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The clinical chemistry revealed significant differences in both sexes with a dose-related trend. A significantly decreased concentration of albumin together with dose–related decreased value of total protein was recorded in both sexes but markedly in male
Urinalysis findings:
no effects observed
Description (incidence and severity):
Changes in urine were observed but statistical analysis of the data revealed no significant differences.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The biometry of organs also detected decreased weight of spleen in both sexes with dose-dependence in females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The application of the test substance caused temporary colouring of the chyme and had negative effect on digestive tract. Infiltration of mucosa and submucosa was recorded in both sexes. Other changes in stomach or forestomach were recorded only at the hi
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the test. No clinically observed signs of toxicity were detected. The animal’s health condition was very good during the whole study.

BODY WEIGHT AND WEIGHT GAIN
A negative effect on body weight was not detected in both sexes.

FOOD CONSUMPTION
A negative effect on dietary intake was not detected in both sexes.

WATER CONSUMPTION
The inspection of water consumption revealed slight decreased consumption in all treated groups of females during the application period. On the contrary during the recovery period the water consumption of treated females was increased. No significant differences were recorded in males.

HAEMATOLOGY
The haematology examination detected significant effect on red blood cells parameters in both sexes. At the end of application period the increased value of hematocrit was recorded at all dose levels but significantly in males of the highest dose level and in females at the lowest and highest dose level. Haemoglobin was insignificantly higher at all treated groups in both sexes. All treated groups of males showed the increased value of mean corpuscular volume and all treated groups of females showed the increased total erythrocyte count. On the contrary after recovery period the value of total erythrocytes count was statistically significantly decreased in both sexes. This decrease was accompanied by significantly increased value of MCV in males and insignificantly increased value of hemoglobin in females. All changed parameters were within historical control range (see Annex 2). These intergroup differences were considered to be the toxicological effect of the application of the test substance.
The increased value of total leucocyte count was found at the middle dose level but in the absence of any other significant changes and of a dose-dependence, the effect are considered to be of no toxicological importance.

CLINICAL CHEMISTRY
The statistical analysis of the biochemical parameters revealed significant differences in both sexes with a dose-related trend. A significantly decreased concentration of albumin together with dose–related decreased value of total protein was recorded in both sexes but markedly in males. An insignificantly increased activity of ALP was recorded at all dose levels in males. The activity of ALT (alanine amonitransferase) was decreased with dose-response relationship in males. Decreased activity of AST (aspartate aminotransferase) in both sexes were recorded at the end of application period. The concentration of cholesterol was decreased at the highest dose of males and inorganic phosphorus was increased in all treated groups of females. After the recovery period, changes were recorded only in females: significantly decreased value of urea, increased concentration of cholesterol and insignificantly decreased activity of AST. Markedly decreased value of creatinine with dose-dependent manner was recorded in both sexes. These changes together with dose-dependent decreased weight of liver can be indications of affection of liver metabolism.

URINALYSIS
Only slightly increased presence of leucocytes in the urine of treated males was detected. In absence of pathological changes in urinary tract, the effects are considered to be of no toxicological importance.

NEUROBEHAVIOUR
The functional observation evidenced no effect of the test substance. All inter- and intra- group differences in behavioural scores (upstanding, emiction, defaecation) and sensory reactivity were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological relevance. No treatment-related changes in functional performance parameters measured (grip strength).

ORGAN WEIGHTS
The biometry of organs detected decreased weight of spleen in both sexes with dose-dependence in females.

GROSS PATHOLOGY
Pathological examination revealed vacuolation of hepatocytes in most of males and females at the highest dose level. These changes are an indication for hepatocellular damage.
The pathology examination revealed black colouring of chyme without colouring of mucous membrane in digestive system at the end of application period. Significant microscopic findings were found in stomach of treated males and females. Infiltration of mucosa and submucosa was recorded in males of all treated groups but markedly at the highest dose level and in females only at the highest dose level. Other changes in stomach or forestomach were recorded only at the highest dose level. Some changes were recorded at the end of recovery period - the effect of the test substance was irreversible. In females at the highest dose level activation of MALT (lymphatic tissue) was recorded in rectum.

HISTOPATHOLOGY: NON-NEOPLASTIC
Metastatic mineralization in kidneys was found in females without dose-dependent manner.
Frequency of other microscopic findings was similar in the treated and control groups and was considered to be of no toxicological importance.

HISTOPATHOLOGY: NEOPLASTIC
No neoplastic findings were recorded by the histopathological examination.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The value of NOAEL (No Observed Adverse Effect Level) for MALES and FEMALES is 400 mg/kg/day. The value was established on the basis of histopathological examination.
Executive summary:

The test substance, Slimes and Sludges, blast furnace and steelmaking, was tested for subacute toxicity using the method B.7. Repeated Dose (28 days) Toxicity (Oral).

Wistar rats of SPF quality were used for testing. The test substance was administered as suspension in olive oil 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 6 males and 6 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 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 - 160, 400, 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.

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

The application of the test substance had no effect on body weight and dietary intake. Water consumption was slightly unbalanced in female.

The haematology examination detected significant irreversible effect on red blood cells parameters in both sexes of all treated groups but markedly at the highest dose level. The increased value of hematocrit and haemoglobin were recorded in both sexes. Males showed the irreversible increased value of mean corpuscular volume and females showed the increased value of total erythrocyte count after application period. After the recovery period the value of total erythrocytes count was the statistically significantly decreased in both sexes. These intergroup differences were considered to be the toxicological effect of the application of the test substance.

The biochemical examination revealed significant differences in both sexes with a dose-related trend. A significantly decreased concentration of albumin together with dose–related decreased value of total protein was recorded in both sexes but markedly in males. An insignificantly increased activity of ALP was recorded at all dose levels in males. The activity of ALT was decreased with dose-response relationship in males. Decreased activity of AST in both sexes was recorded at the end of application period. Markedly decreased value of creatinine with dose-dependent manner was recorded in both sexes. The concentration of cholesterol was decreased at the highest dose of males and inorganic phosphorus was increased in all treated groups of females. After the recovery period, changes were recorded only in females: significantly decreased value of urea, increased concentration of cholesterol and insignificantly decreased activity of AST. These changes together with dose-dependent decreased weight of liver can be indications of affection of liver metabolism. Pathological examination revealed vacuolation of hepatocytes in most of males and females at the highest dose level. These changes are an indication for hepatocellular damage.

The biometry of organs also detected decreased weight of spleen in both sexes with dose-dependence in females.

The application of the test substance caused temporary colouring of the chyme and had negative effect on digestive tract. Infiltration of mucosa and submucosa was recorded in both sexes. Other changes in stomach or forestomach were recorded only at the highest dose level. Some changes were recorded at the end of recovery period - the effect of the test substance was irreversible. In females at the highest dose level activation of MALT (lymphatic tissue) was recorded in rectum.

Frequence of other microscopic findings was minimal or similar in the treated and control groups and was considered to be of no toxicological importance.        

No neoplastic findings were recorded by the histopathological examination.