Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9.3.-17.10.2011
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:
2011
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:
no
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): Tar, brown-coal, low-temp.
- Substance type: technical product - UVCB substance
- Physical state: viscous liquid
- Lot/batch No.: T201E
- Expiration date of the lot/batch: 7.7.2011
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in closed vessel in dry room at the temperature bellow 25ºC in dark.

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
- Age at study initiation: 6-7 weeks
- Fasting period before study: none
- Housing: Animals were housed in polysulfone 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: 9. 3. 2011
Start of administration: 16. -19. 3. 2011
End of administration: 13. – 16. 6. 2011
Clinical examinations: main groups 9. 3. – 16. 6. 2011, satellite groups 9. 3. – 12. 7. 2011
Urinalysis: main groupS 13. – 16. 6. 2011, satellite groups 11. – 12. 7. 2011
Blood taking and necropsies: main groups 14. – 17. 6. 2011, satellite groups 12. – 13. 7. 2011

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.: 4874601
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 (treatment and control group), 6 females and 6 males per group (control satellite and satellite group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen on the basis of results of the 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 group

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: Yes
- 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: during the first week of application and at the end of administration period and recovery period
- Dose groups that were examined: control and the hoghest 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
- Parameters were examined: Total erythrocyte count, Mean corpuscular volume, Haematocrit, Haemoglobin concentration, Total leucocyte count, Total platelets count, Partial thromboplastin time, Prothrombin time, Fibrinogen, Granulocytes, Lymphocytes, Monocytes

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
- Parameters were examined: Glucose, Cholesterol total, Urea, Bilirubin total, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Calcium, Phosphorus, Protein total, Protein albumin, Creatinine, Sodium, Potassium, Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: 90st (main group); 118th (satellite group) day of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters were examined: Volume, Colour, Cloud, Odour, Glucose, Protein, Bilirubin, Urobilinogen, pH, Specific gravity, Blood, Ketones, Nitrite, Leucocytes

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
Complete histological examination (it means examination of all collected organs) was done only in animals of control group and of the highest dose level and in satellite animals. In animals of the low dose level and the middle dose levels the histopathological examination was performed only in those organs where histological changes in the highest dose level or in control were found out (those organs are marked by asterisk* in the table No.5).
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 performed for the results of haematology, blood chemistry, urine pH and volume, biometry of organs and necropsy 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
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during all the study. CLinical changes were recorded during the application period at all dose levels of males and females but markedly at the highest dose level - salivation, disquiet, apathy, changed motility or movement, abdominal or gibbous position with blink eyes. No changes were observed during recovery period.

BODY WEIGHT AND WEIGHT GAIN
Test substance caused decrease of growth increments in both sexes. It was connected with damage of liver metabolism and it was confirmed by the histopathological examination of liver – damage to liver cells: macrovesicular steatosis (mostly with centrolobular localization) and other vacuolation of hepatocytes, single cell necrosis - apoptosis, focal necrosis and presence of hyaline bodies. These irreversible toxicologically important changes were recorded at all treated groups of males and in females at the middle and highest dose levels.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption of treated males and females was lower than at the control groups.

FOOD EFFICIENCY
Markedly decreased conversion was recorded at the highest dose level.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Water consumptions of treated males at the lowest and middle dose levels were lower than in the control group. At the highest dose level the water consumption was markedly higher than in control males. During recovery period the water consumption of treated males was decreased in comparison with the control group.

HAEMATOLOGY
Haematological examination revealed marked symptoms of anaemia in treated males and females. Total erythrocyte count, MCV, concentration of haemoglobin and haematocrit were dose-dependently decreased in both sexes with statistical significance at all dose levels of males and at the middle and highest dose levels of females. These changes were not fully reversible. Anaemia may be secondary event to liver damage. Parameters of blood coagulation were statistically significantly altered in males at the middle and highest dose level and all dose levels of females together with decreased value of platelets count in females at the highest dose level. These changes were not fully reversible.

CLINICAL CHEMISTRY
Biochemical examination showed significant damage of liver at the middle and highest dose level: increased values of cholesterol, total protein (inc. albumin) and changed activity of liver enzymes.
Biochemical examination revealed negative influence of the test substance treatment on kidneys. Increased value of creatinine in blood was recorded in both sexes. Toxicologically important disbalance in ions (sodium, chloride) and minerals (phosphorus and calcium) were measured in both sexes, markedly it occurred at the middle and highest dose levels. These changes were irreversible – statistically significantly differences were recorded also after recovery period.
Increased concentration of glucose in blood related probably to total stress of organism.

URINALYSIS
Urinalysis revealed irreversibly changes with toxicological importance in all groups of treated males and in females at the middle and highest dose levels - decreased pH, presence of protein and leukocytes.

ORGAN WEIGHTS
Treated rats displayed increased weight of liver. Weight of liver was dose–dependently increased in both sexes.
The weight of kidneys was significantly irreversibly increased in males and females at the highest dose level.
At the highest dose level the significantly increased absolute and relative weight of some organs (heart, brain) without presence of macroscopic changes probably related with markedly decreased body weight of animals.
The decreased organs weights of genital organs (prostate gland, epididymides, testes, uterus, ovaries) showed negative irreversible atrophic effect of the test substance treatment in both sexes at the highest dose level.

GROSS PATHOLOGY
In liver of treated males and females marked structure, changed consistence or surface, changed colour (light), enlarged organ and haemorrhages were observed. Reduced prostate gland, and reduced seminal vesicles were registered in males at the highest level. Haemorrhage mesenteric lymph nodes were detected in males and females at the middle and the highest level. Reduced thymus with changed colour was observed in both sexes at the highest level. Changed colour (ochre) of thyroid gland was found in both sexes at the highest level.
Cyst around ovaries and reduced ovaries were found in females at the highest level.

HISTOPATHOLOGY: NON-NEOPLASTIC
The histopathological examination of liver – damage to liver cells: macrovesicular steatosis (mostly with centrolobular localization) and other vacuolation of hepatocytes, single cell necrosis - apoptosis, focal necrosis and presence of hyaline bodies. These irreversible toxicologically important changes were recorded at all treated groups of males and in females at the middle and highest dose levels.
Histopathologic examination of prostate gland and seminal vesicles revealed its atrophy of different degree in most of males at this dose level caused by the test substance administration.
Histopathological examination of thyroid gland found higher frequency of retracted colloid in follicles which was more basophilic in comparison to control animals in treated animals of all dose levels in males and at the middle and highest dose level in females including recovery rats. The cause of this finding is not clear but its relation to the test substance treatment is probable. In females presence of cysts in thyroid gland was recorded only at the middle and highest dose level and in recovery females and this affection was accompanied by the test substance treatment.
Treatment related histopathological finding in kidneys was recorded only in females at the highest dose level – initial glomerulonephrosis and presence of hyaline cats.
In females reduced atrophic ovaries with presence of Sertoli-like tubules were observed in most of animals. Atrophic changes caused an inhibition of oestral cycle in these animals. Dillatation of uterus and accompanied hydrometra (sign of normal physiological function of reproductive organs) was not recorded in any females at the highest dose level.
Abdominal lymph nodes were haemorrhagic with accompanied erythrocytosis and presence of hemosiderin, thymus was reduced and atrophied. Increased presence of rubiginous pigment (probably haemosiderin) in red pulp of spleen of females at the highest dose level was recorded together with increased extramedullary haematopoiesis.

HISTOPATHOLOGY: NEOPLASTIC
Well-differentiated squamous cell carcinoma was revealed in the lungs of one male from the high dose group. This tumor is reported in rats (Greaves, P.:Histopathology of preclinical toxicity studies. Elsevier, Amsterdam, 2000) and its relation to the test substance administered could not be unambiguously excluded.
The microscopical damage to liver cells: macrovesicular steatosis (mostly with centrolobular localization) and other vacuolation of hepatocytes is categorized as preneoplastic lesions.

HISTORICAL CONTROL DATA
Source of data: haematological values and values of biochemical examination of control animals from seven 90-day oral toxicity studies performed during the 2 years.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The value was established mainly on the basis of changes in red blood cell component, biochemical parameters related to kidney function and histopathology and function of the liver.
Dose descriptor:
NOAEL
Effect level:
< 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The value was established mainly on the basis of changes in red blood cell component, biochemical parameters related to kidney function and histopathology and function of the liver.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The value of LOAEL (Lowest Observed Adverse Effect Level) was established as 50 mg/kg/day.
The value of NOAEL (No Observed Adverse Effect Level) is lower than 50 mg/kg/day.
The values were established mainly on the basis of changes in red blood cell component, biochemical parameters related to kidney function and histopathology and function of the liver. The effects of test substance to the liver were of major significance.
Executive summary:

Introduction

   The test substance,Tar, brown-coal, low-temp.,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 theOECD Test Guideline No. 408:Repeated Dose 90-day Oral Toxicity Studyin Rodents, Adopted 21stSeptember 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 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 the 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

    No mortality occurred after test substance treatment. Ophthalmoscopic examination revealed no changes in treated groups of animals.

   The test substance had negative effect on growth increments in both sexes. It was connected with damage of liver metabolism and it was confirmed by the histopathological examination of liver – damage to liver cells. These irreversible changes were recorded at all treated groups of males and in females at the middle and highest dose levels. Weight of liver was dose-dependently increased in both sexes. Biochemical examination also showed significantdamage of liver at the middle and highest dose level: increased values of cholesterol, total protein (inc. albumin) and changed activity of liver enzymes. Observed anaemia may be secondary to liver damage. Parameters of blood coagulation were statistically significantly altered in males at the middle and highest dose level and all dose levels of females. These changes were not fully reversible.

   Biochemical examination revealed negative irreversible influence of the test substance treatment also on kidneys - increased value of creatinine in both sexes, disbalance in ions (sodium, chloride) andminerals (phosphorus and calcium) were measured in both sexes, markedly it occurred at the middle and highest dose levels. The weight of kidneys was significantly irreversiblyincreased in both sexes at the highest dose level. Urinalysis revealed changes with toxicological importance. Treatment related histopathological findings in kidneys were recorded only in females at the highest dose level – initial glomerulonephrosis and presence of hyaline casts.

   Significantly increased concentration of glucose in blood is relatedprobably to total stress of organism.

   The macroscopic findings in genital organs together with decreased organ weights showed negative irreversible atrophic effect of the test substance treatment in both sexes at the highest dose level. Atrophic changes caused an inhibition of oestral cycle in females.

   Thyroid gland of part of males at the middle and highest dose levels and females at the highest showed highest frequency of retracted colloid in follicles which was more basophilic in comparison to control animals. The cause of this finding is not clear but its relation to the test substance treatment is probable. In females presence of cysts in thyroid gland was recorded only at the middle and highest dose level and in recovery females and this affection was accompanied by the test substance treatment.

   Hematopoietic and lymphatic organs showed changes with toxicological significance. Abdominal lymph nodes were haemorrhagic with accompanied erythrocytosis and presence of hemosiderin, thymus was reduced and atrophied. Increased presence of rubiginous pigment (probably haemosiderin) in red pulp of spleen of females at the highest dose level was recorded together with increasedextramedullary haematopoiesis.

  Well-differentiated squamous cell carcinoma was revealed in the lungs of one male from the high dose group. This tumor is reported in ratsand its relation to the test substance administered could not be unambiguously excluded.