Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12.1.2010-2.8.2010
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:
Dust, steelmaking
EC Number:
266-005-7
EC Name:
Dust, steelmaking
Cas Number:
65996-72-7
IUPAC Name:
Dust steelmaking
Details on test material:
- Physical state: solid
- Composition of test material, percentage of components: Fe total 57.64% (mainly as oxides), CaO 8.89%, Zn 4.16%, MgO 3.64%, C 0.69%, SiO2 1.56%, Mn 0.57 %, K2O 0.281%, Na2O 0.251%, Al2O3 0.18%
- Lot/batch No.: 21.10.2009
- Expiration date of the lot/batch: 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., Koleč u Kladna, Czech Republic (SPF breeding)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: cca 206-235 g males; cca 154-174 g females
- Housing: plastic cage (40×25×20 cm) containing sterilised clean shavings of soft woods
- Diet (e.g. ad libitum): peleted diet for rats and mice ST 1 BERGMAN (producer Mill Kocanda, Jesenice by Prag)
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 6 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: This suspension (test substance suspension in olive oil) was mixed for 10 minutes by magnetic stirrer. The application form was prepared daily just before administration.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily just before administration

VEHICLE
- Concentration in vehicle: The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1ml/100 g of body weight.
- Amount of vehicle (if gavage): The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1ml/100 g of body weight.
- Lot/batch no. (if required): 4726901
- Purity: pharmaceutical quality
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
Remarks:
Doses / Concentrations:
120 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg/day
Basis:
actual ingested
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
- Post-exposure recovery period in satellite groups: on the basis of range-finding test results
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 (a part of software 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:
effects observed, treatment-related
Description (incidence and severity):
see Details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
see Details on results
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Changes were observed but effect was negligible.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Changes were observed but effect was negligible.
Food efficiency:
no effects observed
Description (incidence and severity):
Changes were observed but effect was negligible.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Changes were observed but effect was negligible.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Changes were observed but effect was negligible.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Changes were observed but effect was negligible.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the test.
No changes were found out at all dose levels during examination of: position of eyelids, tonic movements, clonic movements, reaction to handling, lacrimation, salivation, emiction, colour of mucosa membranes, vocalisation and other activity.
The reddish-brown secretion around eye in one male at the dose level 120 mg/kg/day, reddish brown secretion around nostrils in one male at the dose level 300 mg/kg/day and in three males at the dose level 750 mg/kg/day were observed. The faeces coloured by test substance was observed at the dose level 750 mg/kg/day in six males in all administration period.
The excrement coloured by test substance was observed at the dose level 750 mg/kg/day in six females in all administration time.

BODY WEIGHT AND WEIGHT GAIN
Average body weight of males and females at all dose levels was relatively well-balanced in comparison with control males for the whole time of study. In treated females mild increase of average body weight in comparison with control was detected - at the dose level 750 mg/kg/day from the 3rd week to the 4th week of study.
Average body weight increment was mildly increased in some animals. Dependence on the dose level was not observed.

FOOD CONSUMPTION AND COMPOUND INTAKE
Average food consumption of males at all dose levels was practically similar in comparison with control males for the whole time of study. The average food consumption of treated females was mildly lower with control females for the whole time of administration period.

WATER CONSUMPTION AND COMPOUND INTAKE
Average water consumption of males of the all dose levels compared to control was higher in the all time of application period (without males of the dose level 300 mg/kg/day in the 3rd and the 4th week). The dose level dependence was not detected. Average water consumption of females at all dose levels was relatively well-balanced in comparison with control females for the whole time of study.

HAEMATOLOGY
Statistically significant difference was detected only in males of the dose level 300 mg/kg/day - increase of value of WBC. Values of WBC in males of the dose level 120 mg/kg/day was increased against control, vice versa of the dose level 750 mg/kg/day was decreased against control. None of parameters were out of our historic control. Statistically significant difference in differential leucocyte count increase of percentage portion of monocytes was found out in females of the dose level 300 mg/kg/day. In females of the all dose levels increased value of APTT (Activated Partial Thromboplastin Time) was detected without dependence on dose level. None of parameters were out of our historic control.

CLINICAL CHEMISTRY
In males in main groups of the all dose levels decreased value of bilirubin was established without dependence on dose level. Decreased value of ALP (Alkaline Phosphate) was detected in males of the dose levels 300 and 750 mg/kg/day. Statistically significant increase of value of calcium and phosphorus were established in females of the dose levels 120 and 750 mg/kg/day. In females of the dose level 300 mg/kg/day calcium and phosphorus were slightly increased without statistical significance. Decreased values of bilirubin and AST (Aspartate Aminotransferase) were detected in females of the all dose levels, decreased values of ALT (Alanine Amonitransferase) and ALP (Alkaline Phosphatase) were detected only of the dose levels 300 and 750 mg/kg/day. Values of other parameters were similar in treated and control animals. None of biochemical parameters were out of historic control.

URINALYSIS
Volume of urine was slightly increased in males of the dose level 750 mg/kg/day and decreased in females of the dose level 120 mg/kg/day. Colour of urine was detected as standard (light yellow) in all treated and control males and females. Values of pH, specific gravity and other parameters of examination of urine of treated males were analogous to values of control animals. Presence of leukocytes in urine was found in all males, slightly increase at the dose level 750 mg/kg/day was determined. Presence of blood in urine was found only in one female of the dose level 300 mg/kg/day.

NEUROBEHAVIOUR
Contact point, reaction to noise, reaction to pain and pupillary reflex of treated animals were same as in the control group. Activity of treated males and females at the all dose levels was slightly higher similar compared to control. Results of emiction and defecation were not same as in the control, but the variation was within the range of the physiological reaction of animals. The values of grip strength of pectoral legs and pelvis legs in treated animals were slightly lower compared to control group – grip power was decreased without dependence on the dose level. All this results were within the range of the physiological reaction of animals.

ORGAN WEIGHTS
There were not noted statistically significant differences of organs weight between treated and control animals.

GROSS PATHOLOGY
In control males and females no macroscopic changes were found out.
In digestive tract the following macroscopic affections were detected: irregular colour of stomach mucosa in three males and three females at the dose 750 mg/kg/day, intestine content coloured by the test substance in all treated males and females, light colour of liver and marked structure of liver in 2 males and 1 female (independent on dose). Dilatation of uterus (pellucid liquid) was recorded in 11 females in treated and control groups. Other macroscopic changes were sporadic (spleen enlarges, lymph node enlarges and fleshy appearance of one lobe in thymus) and were not depend on dose.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological changes or macroscopic findings were found in the heart, kidneys, liver, stomach and thymus. These target organs were examined in medium and low dose group too. Microscopical findings were diagnosed in females very sporadically and were not depend on dose.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No neoplastic findings were recorded by the histopathological examination.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The value was established on the basis of haematology and biochemistry examination.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Clinically observed colouring of stools by the test material in all treated animals was detected. In all animals of the main groups of the highest dose level the colouration of stomach contents by the test substance, in all treated animals (males and females) of all dose level the colouration of large intestine contents was recorded during necropsy. This change was found at the end of application period, at the end of survival period it was not found. The pathology examination revealed black colouring of chyme without colouring and others changes of mucosa membrane in all digestive systems.  

The test substance after 28-day oral application caused irreversible increase of food conversion at the all dose levels without effect on body weight and dietary intake. The animal’s health condition was very good during the whole study and functional observation evidenced no toxic effect of the test substance. 

The haematology examination detected significant irreversible effect on white blood cells parameters in males at the middle dose level (increase of value of total leukocyte count). The irreversible increase of value of total platelet count at the all dose levels in males and decrease in middle level in females were recorded in both sexes.

The biochemical examination revealed significant differences in both sexes. The significantly increased values of calcium and phosphorus ions were recorded in both sexes but markedly in females. These changes of values were delayed (statistically significant increase in satellite males). An insignificantly decreased activity of AST (Aspartate Aminotransferase), ALT (Alanine Amonitransferase), ALP (Alkaline Phosphatase) at the highest dose was recorded in both sexes, decreased of concentration of bilirubin was recorded at all dose levels in both sexes too.

In the urinalysis increase of urine volume in males and on the contrary decrease of urine volume in females were found. 

The biometry of organs also detected changes of weight of liver, kidneys and thymus in both sexes. Mild increase (statistically not significant) of relative weight of the liver and the kidneys at the middle and the highest dose levels in males were found out. On the contrary decrease of relative weight of the liver and the kidneys was found out at all treated females, without dependence on dose level. Increase of relative weight of thymus was detected in both sexes at the highest dose level. Relative weights of other organs of treated males and females were respectively balanced with weights of control animals.

The pathology examination had show, that application of the test substance caused temporary colouring of the chyme without negative effect on digestive tract. The frequency of all 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.

Applicant's summary and conclusion

Conclusions:
The value of NOAEL (No Observed Adverse Effect Level) for MALES and FEMALES is 300 mg/kg/day. The value was established on the basis of haematology and biochemistry examination.
Overall assessment of results showed, that the test substance, Dust, steelmaking after 28-day oral application caused statistically significant increase of value of calcium and phosphorus ions in females and statistically significant decrease of value of calcium and phosphorus ions in satellite males. The observed changes of haematological parameters - irreversible increase of value of total leukocyte count in males, irreversible increase of value of platelet count in males, decrease in females and statistically significant increase of values of prothrombin time in males with delayed effect were considered as adverse.
Executive summary:

The test substance, Dust, steelmaking, was tested for subacute toxicity using the EU method B.7. Repeated Dose (28-days) Toxicity (Oral), Directive 96/54/EC, published in OJ L 248, 1996.

Wistar rats of SPF quality were used for testing. The test substance was administered as suspension 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 6 males and 6 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 120, 300, 750 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 (750 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.

The stability and the homogeneity of the application form in olive oil were not determined. The concentrations of suspension at all dose levels were adjusted to ensure the administered volume of 1 ml per 100 g of body weight. 

Doses for the main study - 120, 300, 750 mg/kg/day were chosen on the basis of results of the study 107/09/7P Dust, steelmaking - Repeated Dose (14 days) Toxicity (Oral) - Dose-range finding experiment. For upper dose level the 750 mg/kg/day (the limit dose level according to the guideline - 1000 mg/kg/day cannot be used in main study) was chosen because in animals administered by the test substance at this dose level death, severe suffering, significant body weight decrement and severe toxic effects were not observed. Two and a half fold interval was used for setting the descending dose levels.

During the 28-day study clinical observation and health status control were performed daily. The body weight and 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.

During control of body weight, food consumption, water consumption, mortality, health condition and during detailed clinical observation, functional observation, and histological examination of treated animals at the dose levels 750, 300 and 120 mg/kg/day no treatment-related effects were detected. There were no unscheduled deaths during the test.

On the contrary the food conversion, general clinical observation, haematological and biochemical parameters, properties of urine, weight of organs and macroscopical appearance of chyme in gastrointestinal tract organs were influenced by administration of the test substance.

The value of NOAEL (No Observed Adverse Effect Level) for MALES and FEMALES is 300 mg/kg/day. The value was established on the basis of haematology and biochemistry examination.

Overall assessment of results showed, that the test substance, Dust, steelmaking after 28-day oral application caused statistically significant increase of value of calcium and phosphorus ions in females and statistically significant decrease of value of calcium and phosphorus ions in satellite males.

The observed changes of haematological parameters - irreversible increase of value of total leucocyte count in males, irreversible increase of value of platelet count in males, decrease in females and statistically significant increase of values of prothrombin time in males with delayed effect were considered as adverse.