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Repeated dose toxicity: oral

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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:
29 Sep 2009 - 10 Jun 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid/colorless, clear
- Storage condition of test material: ambient (room temperature)/ under light exclusion

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Age at study initiation: 41-43 days (males/females)
- Weight at study initiation: males: approx. 170g; females: approx. 135 g
- Housing: 5 animals per cage (H-Temp polysulfonate) with dust-free bedding
- Diet: ad libitum, ground Kliba maintenance diet
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a solution. For preparation, the appropriate amount of test substance was weighed out depending on the desired concentration and filled up with the vehicle (corn oil). The preparations were homogenized using a magnetic stirrer. The testsubstance preparations were prepared weekly.

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The stability of the test substance in corn oil at room temperature for a period of 7 days was proven before the start of the administration period (Study No.: 01Y0356/098022). omogeneity analyses of the test-substance preparations were performed in samples of all concentrations at the start of the administration period. These samples also served for concentration control analyses.
The formulations were shown to be homogeneous and the concentrations under the conditions of administrations were found within the range of 87.8 and 105.8% of the nominal concentrations.
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
males & females
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
males & females
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
females
No. of animals per sex per dose:
5/sex/group plus additional 5/sex in control and high-dose groups for a 2-week recovery period
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 2 weeks for selected male and female animals of the control and high-dose groups

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. All animals were checked daily for any abnormal clinically signs before, up to 1 hour and about 3-4 hours after the administration. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus,, feces (appearance/consistency), urine, pupil size.

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration and the recovery periods the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION: Yes
Individual food consumption was determined weekly over a period of 1 day and calculated as mean food consumption grams per animal and day.

WATER CONSUMPTION: Yes
Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.

CLINICAL PATHOLOGY: Yes
Blood was taken from the retroorbital venous plexus in the morning from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. For urinalysis (main groups only) the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence The assays of blood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the International System (SI). The following examinations were carried out in all animals per test group and sex at the end of the administration period and in the remaining animals at the end of the recovery period (except for enzymes and urinalysis).

HEMATOLOGY:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes, Prothrombin time
(Hepato Quick’s test; HQT). Furthermore, blood smears were prepared and stained according to Wright but were not evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument.

CLINICAL CHEMISTRY:
Parameters analyzed: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea, Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG), Bile acids (BILE)

URINALYSIS:
Parameters analyzed: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Specific Gravity, Sediment, Color, turbidity, Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration (main groups) and the recovery period starting at about 10.00 h. At least one hour before the start of the FOB the animals were transferred singly to polycarbonate cages. Drinking water was provided ad libitum whereas no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: posture, tremor, convulsions, abnormal movements, impairment of gait, other findings.
Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait,, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes.
Sensorimotor Tests/Reflexes: The animals were removed from the open field and subjected to following sensorimotor or reflex tests: approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.
Motor activity measurements: The motor activity (MA) measurement was carried out on all individual animals at the end of the administration (main groups) and the recovery period (in a randomized sequence on each examination day). The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in cages for the time of measurement. Four beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the cages was selected at random. The first measurement of the main groups was started at about 12:45 h and the second measurement of the main groups was started at about 14:00 h. The measurement of the recovery groups was started at about 14:00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles with coagulation glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix.

HISTOPATHOLOGY: Yes
The immunorelevant organs and tissues were evaluated according to the following parameters:
Thymus:
• Increased/decreased grade of cortico-medullar ratio (related only to area)
• Increase of starry sky cells
• Changes of cellular density in the cortex
• Changes of cellular density in the medulla
Spleen:
• Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp
• Altered cellular composition of follicles
• Altered number of germinal centers
Lymph nodes (mesenteric and axillary lymph nodes):
• Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla
• Altered cellular composition of paracortex
• Altered number of germinal centers
• Hyperplasia of high endothelial venules
Peyer's patches (of the jejunum):
• Changes of the cellularity of follicles (including mantle zone and germinal centers)
• Changes of the cellularity of interfollicular area
Bone marrow:
• Changes of the cellularity
• Changes of the myeloid/erythroid ratio
Whenever the histopathologic evaluation of the immunorelevant organs and tissues did not reveal a morphologic alteration of these items and/or whenever no other pathologic finding was noted, these organs were diagnosed as "no abnormalities detected”.
Special attention was given for the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina.
Statistics:
Body weight: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
Feces, FOB parameters, Clinical pathology parameters, urine volume, urine specific gravity, Clinical weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Urinalysis, except color, turbidity, volume and specific gravity: Pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation after treatment was seen in all animals of test group 3 (750/1000 mg/kg bw/d), test group 2 (300 mg/kg bw/d) and in all males and 3 females of test group 1 (100 mg/kg bw/d). This finding was observed on day 8 for the first time and afterwards on several days of the study. No salivation was seen in the recovery period.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely within the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight change were significantly lower in males of test group 3 (750 mg/kg bw/d) on day 7 (body weight: -7.2%; body weight change: -33.0%).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was increased in both sexes of test groups 2 (300 mg/kg bw/d) and 3 (750/1000 mg/kg bw/d) during the administration period. No changes in food consumption were seen in the recovery period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, overt changes in water consumption were observed.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among hematological parameters were measured. All altered hematology parameter values were regarded as being incidental and not treatment-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were measured. All observed alterations among clinical chemistry parameters were regarded as being incidental and not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalyses parameters were measured.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Home cage observations: No test substance-related findings were observed. Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, most observations were considered to have been incidental.
Open field observations: Slight closure eyelid(s) (palpebral closure) was observed in 4 males and 7 females of test group 3 (750/1000 mg/kg bw/d) on study day 23/24. This finding was assessed as being related to treatment. However, no test substance-related findings were observed after recovery.
Sensorimotor tests/reflexes: On study day 23/24, retarded adaptation of the pupil to light (pupillary reflex) was seen in 1 male and 3 females of test group 3 (750/1000 mg/kg bw/d). In addition, no adaptation of the pupil to light (pupils permanently dilated) was observed for 7 females of test group 3 (1000 mg/kg bw/d). Two female animals of test group 3 (1000 mg/kg bw/d) showed very frequent vocalizations when touched. Both types of findings were assessed as being related to dosing. However, no test substance-related findings were observed after recovery.
Quantitative parameters: On study day 23/24 animals of the test group 3 (750/1000 mg/kg bw/d) showed reduced rearing by -37% (males) and significantly reduced rearing -45.1% (females). A relation to dosing was assumed. On study day 41 the grip strength of forelimbs was increased in females in the test group 3 (1000 mg/kg bw/d) up to 17.8%. This finding was assessed as being incidental as grip strength of hindlimbs and foot splay test did not reveal any differnces.
Motor activity measurement: The single interval number 1 was significantly increased in female animals of test group 2 (300 mg/kg bw/d) and decreased in female animals of test group 3 (1000 mg/kg bw/d) on study day 24. The single interval number 6 was significantly decreased in male animals of test group 3 (750 mg/kg bw/d) on study day 23. Furthermore the single interval number 8 was significantly decreased in female animals of test groups 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d) on study day 24. On study day 41, the single interval number 12 was significantly increased in female animals of test group 3 (1000 mg/kg bw/d). These findings were assessed as being incidental and not related to treatment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute weights: A statistically significant increase of the mean absolute liver weight in females of test group 3 (1000 mg/kg bw/d) was regarded as treatment-related. A significant decrease of the ovaries weight was observed in females of test group 2 (300 mg/kg bw/d). This finding occurred without dose-relationship and was therefore considered to be incidental. All other mean absolute weight parameters did not show relevant differences compared to the control group and were regarded to be within the normal range of test animals of that age. In the recovery group, mean absolute organ weights in test groups 1-3 did not show relevant changes compared to the control group.
Relative weights: A statistically significant increase of the spleen weight was noted in males of test group 3 (750 mg/kg bw/d). Since no histopathological findings were observed in the spleen of males of test group 3 (750 mg/kg bw/d), this increase in the relative weight of the spleen was not considered to be treatment-related.The significant increase in the relative weight of the liver in females of test group 3 (1000 mg/kg bw/d) was regarded as treatment-related. The significant decrease of the ovaries weight in females of test group 2 (300 mg/kg bw/d) occurred without dose-relationship and was therefore considered to be incidental. All other mean relative weight parameters of the treated animals did not show relevant differences compared to the control groups. In the recovery group, the brain weight in males of test group 3 (750 mg/kg bw/d) revealed a significant increase (117%, p ≤ 0.01) compared to controls (set to 100%). This increase was considered to be in relation with a non-significant decrease of the terminal body weight. All other mean relative weight parameters did not show relevant differences compared to the control group.
See also tables below.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All gross findings observed in test animals occurred singularly. They were considered to be spontaneous in origin and were not related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related histopathological findings were observed in test animals. All findings noted were either single observations or were biologically equally distributed between control and treated rats. All of them were considered to be incidental and/or spontaneous in origin.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: eye findings (mydriasis) at dose levels of 750 mg/kg bw/d (males) and 1000 mg/kg bw/d (females).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

ORGAN WEIGHTS

Compared to controls (set to 100%) following values (expressed in %) were significantly increased:

Male animals Female Animal
Test group 1 2 3 1 2 3
Liver 98% 101% 117%*
Ovaries 94% 82%* 92%

* : p ≤ 0.05; **: p ≤ 0.01

Compared to controls (set to 100%) following values (expressed in %) were significantly increased:

Male animals Female Animal
Test group 1 2 3 1 2 3
Spleen 100% 111% 128%*
Liver 99% 100% 108%
Ovaries 94% 81%* 84%

* : p ≤ 0.05; **: p ≤ 0.01

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, the no observed adverse effect level (NOAEL) of the test substance was 300 mg/kg bw/d for male and female Wistar rats taking into account the eye findings (mydriasis) at dose levels of 750 mg/kg bw/d (males) and 1000 mg/kg bw/d (females). Increased liver weights in females were assessed as adaptive and non-adverse as no histopathological findings were observed. Salivation was considered to be related to either the bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.
Executive summary:

The test substance was administered daily by gavage to male and female Wistar rats at dose levels of 0 (test group 0; corn oil served as vehicle), 100 (test group 1), 300 (test group 2) and 750 (test group 3, males)/1000 (test group 3, females) mg/kg body weight/day (mg/kg bw/d) over a period of 28 days. Control and test group 3 (750 mg/kg bw/d [males]/1000 mg/kg bw/d [females]) consisted of each 10 animals per sex, whereas test groups 1 (100 mg/kg bw/d) and 2 (300 mg/kg bw/d) consisted of each 5 animals per sex. After 4 weeks of treatment 5 animals per sex of all test groups were sacrificed (main groups). The remaining 5 animals per sex of control and test group 3 (750 mg/kg bw/d [males]/1000 mg/kg bw/d [females]) were maintained for another 14 days without administration of the test substance (recovery groups).

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity and mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Additionally, a functional observational battery (FOB) as well as measurement of motor activity (MA) was carried out at the end of the administration as well as the end of the recovery period. Clinicochemical and hematological examinations were performed towards the end of the administration and the end of the recovery period. Urinalyses were only done for the main groups. All animals were assessed by gross pathology, followed by histopathological examinations.

The following test substance-related, adverse findings were noted in test group 3 during clinical examinations: no adaptation of the pupil to light in 7 female animals, retarded adaptation of the pupil to light in 1 male and 3 female animals, slight closure of eyelid(s) in 4 males and 7 females. During clinical pathology, no test substance-related, adverse findings were observed. No test substance-related, adverse findings were observed during pathology. Increased liver weights in females were assessed as adaptive and non-adverse as no histopathological findings were observed. Adverse findings were confined to the high-dose group, only and displayed predominantly as effect on pupil adaptation to light. Thus, under the conditions of the present study, the no observed adverse effect level (NOAEL) of th test article was 300 mg/kg bw/d for male and female Wistar rats taking into account the eye findings (e.g. mydriasis) at dose levels of 750 mg/kg bw/d (males) and 1000 mg/kg bw/d (females).