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Toxicological information

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:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study according to OECD guideline 408 (adopted 1998). Minor restrictions: ophthalmological examination not specifically stated in the document but weekly investigation of eyes concerning mydriasis, miosis, and exophthalmos as well as histopathology of eyes after termination.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
(Harlan Laboratories Ltd.)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Biofert Plusz
Details are presented in "Confidential details on test material"

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Laboratory Animal Services, Füllinsdorf / Switzerland
- Age at delivery: 7 weeks
- Weight at acclimatisation: males 175 g to 200 g (mean 187 g), females: 139 g to 156 g (mean 146 g)
- Fasting period before study: no
- Housing: In groups of five in Makrolon type-4 cages
- certified Diet ad libitum
- Tap water ad libitum
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used.

ENVIRONMENTAL CONDITIONS
according to Guideline

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Bidistilled water
Details on oral exposure:
The dose formulations were prepared weekly. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. The dose formulations were stored refrigerated in glass beakers (2 - 8 °C). Dose Volume: 10 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Results of the test item dose formulations analyses: range of 93.2% to 121.5%. For 33 of 36 samples, the required content limit of ±20% with reference to the nominal concentration was fulfilled. (3 samples exceeded the upper range). All were found to be homogeneous, and all were found to be stable for four hours at room temperature and for seven days when refrigerated (2 - 8 °C).
Duration of treatment / exposure:
91 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
other: related to dry mass (analytical verification)
Remarks:
Doses / Concentrations:
0, 356, 1068, 3559 mg/kg bw/day
Basis:
other: related to liquid test item
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: related to results in subacute dose-finding study; high dose level corresponds to the limit dose recommended in OECD Guideline 408.
- Rationale for animal assignment: randomisation
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: no
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs once before commencement of administration as well as twice daily on days 1 to 3 and once daily on days 4 - 91 (treatment period).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during the acclimatization and treatment periods and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption: The food consumption was recorded once during the acclimatization period and weekly thereafter

FOOD EFFICIENCY:
- The mean absolute food consumption and the mean relative food consumption of the treated rats compared with controls.

OPHTHALMOSCOPIC EXAMINATION: No, however, once weekly investigation of mydriasis, miosis, and exophthalmos.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once after 13 weeks of exposure
- Anaesthetic used for blood collection: Yes (from the retro-orbital plexus from all animals under light isoflurane anesthesia)
- Animals fasted: Yes (18 h)
- How many animals: all
- Parameters:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index (low, medium,
high fluorescence)
Leukocyte count, total
Differential leukocyte count:
Neutrophils
Eosinophils
Basophils
Lymphocytes
Monocytes
Large unstained cells
Platelet count
Methemoglobin
Heinz bodies (slides prepared but not evaluated)
Coagulation
Prothrombin time (= Thromboplastin time); Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see hematology
- Animals fasted: Yes (18 h)
- How many animals: all
- Parameters:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Alkaline phosphatase
Phospholipids
Gamma-glutamyl-ransferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the 18 hours fasting period (see hematology) into a specimen vial, using a metabolism cage.
- Parameters:
Urine volume (18 hour)
Specific gravity (relative density)
Color
Appearance
pH value
Nitrite
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Erythrocytes
Leukocytes

DETAILED BEHAVIORAL OBSERVATIONS (Once weekly, alle rats)
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 12) thereafter.
Parameter:
Piloerection
Salivation
Hunched posture
Ataxia
Tremor/twitching
Prostration
Circling
Spasm
Hyperactivity
Somnolence
Increased exploration
Reduced grooming
Vocalisation
Dyspnea
Tachypnea
Bradypnea

Refexes:
Blink
Pinna
Iridic light reflex
Push-off (hind leg)
Pain response
Startle/hearing
Righting reflex

Lacrimation
Limbs cyanotic
Mydriasis
Miosis
Exophthalmos
Reduced muscle tone

FUNCTIONAL OBSERVATION BATTERY
During week 13 (all animals), relevant parameters: Grip strength and locomotor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ wet weights determined of all organ recommended in OECD408 (liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, thymus, spleen, brain, heart)

HISTOPATHOLOGY: Yes (all organ recommended in OECD408)
Adrenal glands
Aorta
Bone (sternum, femur including joint)
Bone marrow (femur)
Brain - including section of medulla/pons, cerebral and cerebellar cortex
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Oesophagus
Eyes w/optic nerve (fixed in Davidson's solution)
Harderian gland (fixed in Davidson's solution)
Heart including auricles
Ileum, with Peyer's patches
Jejunum with Peyer's patches
Kidneys
Larynx
Lacrimal gland, exorbital
Liver
Lungs, filled w/formalin at necropsy
Lymph nodes – mesenteric and mandibular
Mammary gland area
Nasal cavity
Ovaries
Pancreas
Pharynx
Pituitary gland
Prostate gland incl. coagulating glands
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland, if possible)
Tongue
Trachea
Urinary bladder, filled w/formalin at necropsy
Uterus with cervix as appropriate
Vagina
All gross lesions
Other examinations:
no
Statistics:
Statistical analysis of body weight, clinical laboratory data, locomotor activity, grip strength, organ weights and ratios as well as macroscopic findings.
- The Dunnett-test (many to one t-test;normal distribution)
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when data could not be assumed to follow a normal distribution
- Fisher's exact-test

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):
not examined
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until scheduled necropsy. There were no clinical signs evident in any test item-treated rat at any time during the study. Detailed clinical observations (weekly): During weeks 1-12, no clinical signs were evident at any dose level and no behavioral changes were detected.
Functional observational battery: During week 13, no clinical signs were evident at any dose level and no behavioral changes were detected. Grip strength: The differences were marginal (mid and high dose males significantly elevated mean hind limb grip strength values) and considered to be within the range of normal biological variation, and of no toxicological relevance.
Locomotor Activity: No test item-related changes in the mean locomotor activity were noted. In males treated with 300 mg/kg/day, a statistically significant reduction (p<0.01) was noted during the 30-40 minute interval. In the absence of similar findings in high dose males, this isolated difference was considered to be incidental.

BODY WEIGHT AND WEIGHT GAIN
On Days 1, 8, 15, 22, 29 and 71, the mean absolute body weights of the males treated with 1000 mg/kg bw/day were significantly elevated (p<0.05) when compared with controls. These differences were considered to result from slightly higher body weights from Day 1 of treatment and were <=6% compared to concurrent control. The body weight gain of males did not show any significant differences from control values.
In females treated with 100 mg/kg bw/day, the mean absolute body weights were significantly higher (p<0.05) on Day 64 of treatment, however, the difference was only 6.1%. No significant effects were detected on absolute body weight at the mid and high dose level. In females treated with 300 mg/kg bw/day, the mean body weight gain was significantly elevated on several occasions (p<0.05 on Days 15, 29, 43, 57 and 64; p<0.01 on Days 8 and 36) but not at day 71 and later. In the absence of similar changes of similar magnitude in females at 1000 mg/kg bw/day (although a statistically significant difference (p<0.05) at day36), these differences in body weight gain were considered to be incidental.

FOOD CONSUMPTION
The mean absolute food consumption and the mean relative food consumption of the test item treated rats compared favorably with those of the controls.

HAEMATOLOGY
In males, statistically elevated red blood cell counts were noted at 1000 mg/kg bw/day (p<0.05), whereas the mean hemoglobin distribution width was reduced (p<0.05). Males treated with 300 mg/kg bw/day had a significantly reduced lymphocyte count (p<0.05) and a significantly lower mean thromboplastin time (p<0.01). In males of the low dose level, the mean relative basophil count was significantly elevated (p<0.05).
In females, significantly elevated hemoglobin was recorded at 1000 mg/kg bw/day (p<0.05) and the mean absolute leukocyte count was significantly elevated (p<0.05; mean absolute elevated basophils (p<0.05), monocytes (p<0.05) and ‘large unstained cells’ (p<0.05). At 300 mg/kg bw/day, the mean relative thromboplastin time was elevated in females (p<0.05) and at 100 mg/kg/day, the hemoglobin distribution width was significantly lower (p<0.05).
All these changes were not considered to be of toxicological relevance. These altered values remained within the ranges of the historical control ranges and/or were not dose related and therefore considered to be incidental.

CLINICAL CHEMISTRY
Also no test item-related changes of toxicological relevance were noted in the clinical biochemistry parameters at any dose level.
In males treated with 1000 mg/kg bw/day, significantly elevated sodium (p<0.01), calcium (p<0.05) levels were found and in the mid dose group males significantly lower (p<0.01) phosphorus levels, however, all values were within the historical control values. Although the albumin level remained within the range of the historical control data, the mean globulin level slightly exceeded it also raising the total protein level slightly above the upper
limit of historical control data. No toxicological relevance was associated with this difference.
Sodium and chloride levels were significantly elevated in high dose females and mid dose females showed elevated blood glucose (p<0.05), triglycerides (p<0.01) and sodium (p<0.05). Low dose females showed marginally elevated potassium (p<0.05). All values remained within the ranges of the historical control values.

URINALYSIS
No test item-related changes were noted in the urinalysis parameters at any dose level. Males treated with 1000 mg/kg bw/day had significantly reduced urinary pH (p<0.05) when compared with the concurrent controls. However, these values were within the historical control range.

ORGAN WEIGHTS
There were no differences of toxicological relevance between the mean absolute or relative organ weights of test item-treated and control animals.

GROSS PATHOLOGY
A small number of typical background findings were noted at necropsy. None of these findings were considered to be of toxicological relevance (stomach foci in one male and one female at 1000 mg/kg bw/day and in two control males, renal pelvis dilatation one male and one female at 1000 mg/kg bw/day and in one control female, foci on the thymus in one male at 300 mg/kg bw/day and 1000 mg/kg bw/day each, a nodule in the body cavity of one male at 1000 mg/kg bw/day and food-like material in the esophagus).

HISTOPATHOLOGY:
The test item produced no histopathological effects. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
other: dry mass of the liquid test item
Sex:
male/female
Basis for effect level:
other: highest dose level tested which corresponds to the limit dose recommended in OECD Guideline 408

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Body weight gain in female rats (in % of absolute body weight)

Means+-standard deviation

Day of treatment

Vehicle control

100 mg/kg bw/day

200 mg/kg bw/day

1000 mg/kg bw/day

1

0

0

0

0

8

7.3+-3.2

8.2+-1.7

12.0+-2.7**

10.0+-2.8

15

16.3+-2.6

17.5+-2.0

20.4+-4.5*

19.5+-3.7

22

22.7+-3.2

22.5+-3.5

27.2+-5.5

25.9+-4.8

29

29.0+-4.1

29.9+-2.9

33.4+-4.4*

30.8+-3.9

36

32.3+-5.2

34.7+-4.1

39.1+-4.9**

37.6+-5.0*

43

36.6+-4.1

38.9+-3.9

43.3+-6.6*

41.3+-6.3

50

41.2+-4.0

42.5+-5.4

46.4+-6.8

45.5+-6.0

57

43.8+-4.6

46.6+-4.2

49.4+-5.0*

46.3+-4.1

64

43.8+-6.0

49.8+-5.0

51.1+-5.6*

49.4+-5.8

71

46.7+-5.1

49.5+-5.3

52.4+-6.8

50.7+-7.5

No significant effects at later time points

*: significant, p<0.05; **: significant, p<0.01

Applicant's summary and conclusion

Conclusions:
In a sub-chronic gavage study according to OECD Guideline 408 in male and female Wistar rats no effects of toxicological relevance were detected even at the high dose level of 1000 mg/kg bw/day (related to dry mass).