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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:
26.07.2009 – 25.03.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Semi Dry Absorption (SDA) Product
- Molecular formula (if other than submission substance): not available
- Molecular weight (if other than submission substance): not available

Composition of test substance:
Calcium sulphate 6.49 %
Calcium sulphite 41.40 %
Calcium carbonate 29.50 %
Calcium hydroxide 2.27 %
Calcium chloride 17.98 %
Calcium fluoride 0.57 %
Oxides (SiO2 + Al2O3 + Fe2O3 + TiO2) sum 0.97 %
Sum of toxic metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn sum < 0.1 %

Batch No.: SDA/0609/Sk
Appearance: White solid powder
Stability / Expiration date: 15 years (06/2024)
- Stability under test conditions: stable
- Storage condition of test material: the substance was stored in PE container at room temperature.
- Other: pH 11 approx. (by contact of application form with universal indicator pH strip moistened with water, strip producer Lach-Ner, s.r.o. Neratovice)

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., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- sex: males and virgin females
- Age at study initiation: 6-7 weeks
- Fasting period before study: no
- Weight at study initiation: At the beginning of the study the weight variation of animals in groups of each sex did not exceed ± 20% of the mean weight.
- Housing: Animals were housed in SPF animal room, 2 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, manufacturer: Ing. Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy; sterilised before using)
Composition of diet: Wheat, Oats, Fish meal powder, Dried snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex. Nutrient content 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%.
- Water: ad libitum (water quality corresponded to Reg. No. 252/2004 Czech Coll. of Law; sterilised before using)
- Acclimation period: 8 days
- Identification: Identification of animals was made by colour marks on fur (system 1 – 10), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance and mark of group.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): a relative humidity of 30-70%
- Air changes (per hr): Animals were housed in controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle.

IN-LIFE DATES:
Study Time Schedule
Test substance delivery: 16.7.2009
Date of animal arrival: 24.8.2009
Start of administration: 2.9. 2009
End of administration: 3.12.2009
Cllinical examination:
main groups 24.8. – 4.12.2009
satell. groups 24.8. – 30.12.2009
Urinalysis:
main groups 30.11. – 3.12.2009
satell. groups 28.– 29.12.2009
Blood taking and necropsies:
main groups 1.– 4.12.2009
satell. groups 29.– 30.12.2009
Histopathological examination: 4.1.2010 – 12.3.2010
Evaluation of results and final report elaboration: 10.1.2009 – 25.3.2010

- Additional Information: The standard pelleted laboratory animal diet are analysed for nutrients (once a year) and bacteriologically examined (every two months) on a regular basis. Results are retained in the CETA archives. Reports of analysis of water (twice a year) are retained in the CETA archives. Results of sterilizer effectivity control (performed once a year) are retained in the CETA archives. Analysis of diet and water and steriliser control, did not reveal any findings that could affect study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1mL per 100 g of body weight.
The application form of the test substance (suspension in olive oil) was prepared daily just before administration. The mixture was mixed for 10 minutes (cca 800 rpm). The vehicle control group was administered by olive oil in the same volume.

VEHICLE
- Olive oil (Olivae oleum raffinatum)
- Batch No.: L 803 142, L46/8 3401
Manufacturer: Dr. Kulich Pharma, s.r.o., Hradec Králové, Czech Republic


Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test substance consists of various oxides insoluble in the application form (olive oil). A suitable analytical method cannot be found for homogeneity and stability testing. Since undissolved particles of the test substance are easily visible in the application form, homogeneity was checked by eye (suspension were mixed for 10 minutes by magnetic stirrer). Stability of the test substance in the application form cannot be verified but there is no indication that a mixture of rigid oxides would be unstable in its solution (in olive oil) for that short time period (1 hour).
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days per week at the same time
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:
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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first application and then weekly

MORTALITY: Yes
- Time schedule: daily

CLINICAL OBSERVATION: Yes
- Time schedule: daily during the administration period

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and immediately before euthanasia

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

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

FUNCTIONAL OBSERVATION: Yes
- Time schedule: at the last week of administration period and recovery period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the 1st week of study and at the last week of administration 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.
Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

Pathological Examination - 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.

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
Description (incidence and severity):
Treatment-related increase of urine volume was recorded markedly in males together with increased water consumption in all treated groups. This was probably due taste irritancy of the test substance, and as no functional disturbance was observed (without histopathological findings in urinary system), to be of no toxicological relevance.
Ophthalmological findings:
no effects observed
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):
see Details on results
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Details on results
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL AND FUNCTIONAL OBSERVATIONS AND MORTALITY
There were no treatment revealed changes.

BODY WEIGHT AND WEIGHT GAIN, FOOD CONSUMPTION
A slight intergroup differences in body weight and food consumption of treated animals was without dose dependence and statistically insignificant.These findings were probably no toxicological relevance.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment revealed changes.

HAEMATOLOGY
Several haematological parameters were significantly affected by treatment at 1000 mg/kg/day but had prolonged effect (was recorded after recovery period. These changes consisted of effect on red blood cells in males and females (increased levels of haemoglobine together with correlated parameters) and effects on clotting parameters in males (increased protrombin time, decreased concentration of fibrinogen and decreased partial thromboplastin time). These changes are an indication for increased erythrocyte turnover. However, no extra-medullary haematopoesis was seen in any organ. Effect on white blood cells (significantly increased level of granulocytes and slight increased leucocyte count) was recorded only in females also after recovery period.

CLINICAL CHEMISTRY
Some clinical biochemistry parameters were significantly affected by treatment at 1000 mg/kg/day. For males, statistically significantly decreased value of urea and decreased activity of AST were observed after application period and decreased concentration of glucose and increased levels of phosphorus and sodium ions were measured after recovery period. On the contrary in females increased concentration calcium after application period and decreased level of total protein and creatinine after recovery period were recorded. In absence of a treatment-related distribution they were considered changes of no toxicological significance.

URINALYSIS
Urinalysis revealed presenting of leucocytes in urine of all treated males and some of treated females. Treatment-related increase of urine volume was recorded markedly in males together with increased water consumption in all treated groups. This was probably due taste irritancy of the test substance, and as no functional disturbance was observed (without histopathological findings in urinary system), to be of no toxicological relevance.

ORGAN WEIGHTS
There were no treatment revealed changes.

HISTOPATHOLOGY NON-NEOPLASTIC
Histopathological examination revealed significant changes only in males at the highest dose level. Affections of mucous membrane in stomach (erosions, haemorrhages or other focal changes) and affections in heart (haemorrhages or myocardititis) were found out after application period and probably correlated with application of the test substance. Males of the lowest and middle groups and all groups of females showed only sporadically changes without toxicological significance.

Examination of focal fatty changes (presence of small or large spherical vacuoles in cytoplasm of hepatocytes) revealed slight changes in distribution in treated males. At all dose levels irregular, peripheral, intermediate, centrolobular or diffuse steatosis in the liver was noted. Control males showed only peripheral steatosis. Although this finding was more common in treated groups, the hepatocellular damage (vacuolation of hepatocytes and dystrophy) was found out only sporadically at the highest dose level.

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

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: The value was established on the basis of haematology, urinalysis and histopathological examination.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: The value was established on the basis of haematology, urinalysis and histopathological examination.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The value of NOAEL (No Observed Adverse Effect Level) for MALES is 400 mg/kg/day. The value was established on the basis of haematology, urinalysis and histopathological examination.
The value of NOAEL (No Observed Adverse Effect Level) for FEMALES is 1000 mg/kg/day.
Executive summary:

Introduction

The test substance, Semi Dry Absorption (SDA) Product, 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 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 90 days. After that animals of main groups were sacrificed and satellite animals were observed for the next 28 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 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 Semi Dry Absorption (SDA) Product to rats by gavage for period of 90 consecutive days at the dose levels 160, 400, 1000 mg/kg/day did not cause any mortality. There were no treatment revealed changes for clinical observation, functional observations, ophthalmoscopic examinations and biometry of organs.

A slight intergroup differences in body weight and food consumption were probably no toxicological relevance.

Several haematological parameters were significantly affected by treatment at 1000 mg/kg/day but had prolongated effect (was recorded after recovery period). These changes consisted of effect on red blood cells in males and females (increased levels of haemoglobine together with correlated parameters) and effects on clotting parameters in males (increased protrombin time and concentration of fibrinogen, decreased partial thromboplastin time). These changes are an indication for increased erythrocyte turnover. However, no extra-medullary haematopoesis was seen in any organ. Effect on white blood cells (significantly increased level of granulocytes and slight increased leukocyte count) was recorded only in females also after recovery period.

Some clinical biochemistry parameters were significantly affected by treatment at 1000 mg/kg/day. For males, statistically significantly decreased value of urea and decreased activity of AST were observed after application period and decreased concentration of glucose and increased levels of phosphorus and sodium ions were measured after recovery period. On the contrary in females increased concentration of calcium after application period and decreased level of total protein and creatinine after recovery period were recorded. In absence of a treatment-related distribution they were considered changes of no toxicological significance.

Urinalysis revealed presence of leucocytes in urine of all treated males and some of treated females. Treatment-related increase of urine volume was recorded markedly in males together with increased water consumption in all treated groups. This was probably due to taste irritancy of the test substance, and as no functional disturbance was observed (without histopathological findings in urinary system), to be of no toxicological relevance.

Histopathological examination revealed significant changes in stomach (erosions, haemorrhages or other focal changes) and in heart (haemorrhages or myocardititis). These affections were found out only in males at the highest dose level after application period and probably correlated with application of the test substance. Males of the lowest and middle groups and all groups of females showed only sporadically changes without toxicological significance.

Examination of focal fatty changes (presence of small or large spherical vacuoles in cytoplasm of hepatocytes) revealed slight changes in distribution in treated males. At all dose levels irregular, peripheral, intermediate, centrolobular or diffuse steatosis in the liver was noted. Control males showed only peripheral steatosis. Although this finding was more common in treated groups, there was hepatocellular damage (vacuolation of hepatocytes and dystrophy) found out only sporadically at the highest dose level.

The value of NOAEL (No Observed Adverse Effect Level) for MALES is 400 mg/kg/day. The value was established on the basis of haematology, urinalysis and histopathological examination.

The value of NOAEL (No Observed Adverse Effect Level) for FEMALES is 1000 mg/kg/day.