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Toxicological information

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:
26 Nov - 24 Dec 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study. The weight of the uterus and ovaries were not recorded.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted Sept 1996
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
adopted July 1995
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. OPPTS (7101), EPA 712-C-00-366 (adopted July 2000)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: powder aggregated into lumps
Details on test material:
- Name of test material (as cited in study report): SPM-N
- Physical state: white lumps with powder
- Lot/batch No.: T-9314
- Expiration date of the lot/batch: 29 Aug 2008
- Storage condition of test material: at room temperature in the dark
- Other: pH 7.6-7.5 (1% in water, indicative range)

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 145-164 g (range, males), 122-146 g (range, females)
- Housing: animals were housed in groups of 5 per sex in Macrolon cages (MIV type, height 18 cm), with sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During overnight activity monitoring animals were housed individually (in MIII type, height 15 cm) with no cage-enrichement.
- Diet: pelleted rodent diet (SM R/M from SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7-21.6
- Humidity (%): 27-81
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Nov 2007 To: 24 Dec 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Solutions (w/w) were prepared daily within 4 h before dosing and were homogenised to visually acceptable levels. Adjustment was made for the specific gravity of the vehicle. The formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at the testing laboratory and on information from the sponsor.
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations were analysed once during the study period to check the accuracy of preparations (all doses), and the stability and homogeneity (highest and lowest dose) of the test substance in the formulations. The formulations were stirred during the sampling. Duplicate samples for the determination of stability were taken at 50% height of the formulation and stored for 5 hours. For the determination of the accuracy and homogeneity, duplicate samples were taken at 90% height, at 50% height and at 10% height of the formulation. The analytical concentration was determined for all the samples by HPLC analysis. The actual concentrations were found to be 97-100% of nominal, which represents an acceptable level of accuracy and homogeneity. In addition the substance formulation propylene glycol was noted as stable for at least 5 hours and homogenous (a relative difference of -0.11 to 5.3% relative to mean concentration).
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150, 1000 mg/kg bw/day
Basis:
other: nominal by gavage
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a range-finding study (project 486463) 3 rats/sex/group were administered 500 and 1000 mg/kg bw/day for 5 days. No mortality was observed and no effect on the body weight or food consumption was noted. In 1 female in the 500 mg/kg bw/day group rales (rattling breath) was noted. The liver and kidney weight were normal and no gross pathological effects were observed. 1000 mg/kg bw/day is the recommended maximum dose according to OECD Guideline 407. Based on the results of the range-finding study, 50, 150 and 1000 mg/kg bw/day was selected as the doses for the main study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality, once daily for detailed clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes. Observations were made outside the home cage in a standard arena.
- Time schedule: once before the first exposure on Day 1, and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: once before the first exposure on Day 1, and thereafter on Day 8, 15, 22 and 28

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption was measured weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No, only a subjective appraisal was maintained during the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy on Day 28, drawn from the retro-orbital sinus
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight for a maximum of 20 h
- How many animals: 5/sex/dose
- Parameters examined: white blood cells, differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes), red blood cells, reticulocytes, red blood cell distribution width, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy on Day 28, drawn from the retro-orbital sinus
- Animals fasted: Yes, overnight for a maximum of 20 h
- How many animals: 5/sex/dose
- Parameters examined: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), protein (total), albumin, bilirubin (total), urea, creatinine, glucose, cholesterol, potassium, sodium, calcium, chloride, inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4 of the study for males and females
- Dose groups that were examined: all dose groups
- Battery of functions tested:
Standard functional observational battery (FOB) including grip strength measurements, auditory response, static righting reflex, pupillary reflex; and motor activity (MA), measured as number of movements per hour over a 12 h period, with a computerised motor activity monitoring system (Pearson Technical Services, Debenham, Stowmarket, England). Each animal was tested individually.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were necropsied and the tissues/organs subjected to macroscopic examination.
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus.

HISTOPATHOLOGY: Yes. Samples of the tissues/organs listed below were collected from all animals and fixed in 10% buffered formalin. Slides of all organs and tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group, and all gross lesions of all animals, were prepared and examined. Slides were stained with hematoxylin and eosin. Tissues mentioned between brackets were not examined as there were no signs of target organ involvement.

Samples collected from: adrenal glands, aorta, brain - cerebellum, mid-brain and cortex, caecum, (cervix), (clitoral gland), colon, duodenum, epididymides, (eyes with optic nerve and Harderian gland), (female mammary gland area), (femur including joint), heart, ileum, jejunum, kidneys, (larynx), (lacrimal gland, exorbital), liver, lung - infused with formalin, lymph nodes - mandibular and mesenteric, (nasopharynx), oesophagus, ovaries, pancreas, Peyer’s patches- jejunum and ileum - if detectable, pituitary gland, (preputial gland), prostate gland, rectum, (salivary glands - mandibular and sublingual), sciatic nerve, (seminal vesicles), (skeletal muscle), (skin), spinal cord - cervical, midthoracic and lumbar, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, (tongue), trachea, urinary bladder, uterus, (vagina), all gross lesions
(tissues mentioned in brackets were not examined as there were no signs of target organ involvement)
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. The exact Fisher-test was applied to frequency data. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: increased red blood cell distribution width, males (non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
150 and 1000 mg/kg bw/day: increased chloride levels (non-adverse), decreased ASAT level, males (non-adverse);
Urinalysis findings:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: plasmacytic hyperplasia of the mandibular lymph nodes; thymus atrophy, females (non-adverse)
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no mortality during the study period. 1/10 males in the low-dose group had alopecia from Day 1-13 and scabs on the shoulder (Day 6-13) and flews (Day 13-21). At the incidence observed, these were considered signs of no toxicological significance. No other clinical signs were observed in the animals.

BODY WEIGHT AND WEIGHT GAIN
The body weight and body weight gain was comparable between the control group and treatment groups during the whole study period.

FOOD CONSUMPTION
The food consumption was comparable between the control group and treatment groups during the whole study period.

HAEMATOLOGY
The red blood cell distribution width (RDW) was significantly increased in the males of the high-dose group (control: 11.6 ± 0.5%; 1000 mg/kg bw/day: 13.8 ± 2.1). This was mainly due to the high level measured in one animal. Moreover, the higher RDW was well within the normal range for rats of this age and strain and occurred in the absence of corroborative haematological changes. Thus this change in RDW is not considered to be a toxicologically relevant effect. In the females of the low-dose group, the level of eosinophils (as percentage of white blood cells) was significantly decreased (control: 1.9 ± 0.7%; 50 mg/kg bw/day: 1.0 ± 0.2%). As no similar effect was observed in any other treatment group, this is considered to be an incidental occurrence.

CLINICAL CHEMISTRY
The chloride level was significantly increased in males and females of the high-dose group (males: control: 103 ± 1 mmol/L, 1000 mg/kg bw/day: 106 ± 2 mmol/L; females: control: 105 ± 0 mmol/L, 1000 mg/kg bw/day: 109 ± 1 mmol/L) and in females in the mid-dose group (control: 105 ± 0 mmol/L, 150 mg/kg bw/day: 108 ± 1 mmol/L). The higher chloride levels were not accompanied by any morphological changes. Taking also account the mild nature of this change, this is not considered to be a toxicologically significant effect.
The ASAT level was decreased in males in the mid- and high-dose groups (control: 76.4 ± 5.6 IU/L, 150 mg/kg bw/day: 62.9 ± 2.1 IU/L, 1000 mg/kg bw/day: 64.0 ± 11.4 IU/L). The females did not show similar decreases and no effects on liver were noted during gross pathology and histopathology examinations. Generally, an opposite and more pronounced change in the ASAT level is expected to indicate possible target organ toxicity. Therefore, the effect is not considered to be toxicologically relevant.
In the high-dose group of males, a significant decrease in the calcium level was noted (control: 2.83 ± 0.04 mmol/L, 1000 mg/kg bw/day: 2.72 ± 0.06 mmol/L. However, the mean calcium level remained in the range considered normal for rats of this strain and age and was thus considered to be of no toxicologically significance. Changes in the levels of inorganic phosphate and glucose in a single low- or mid-dose group occurred in the absence of a dose-related trend and are considered to be incidental occurrences.

NEUROBEHAVIOUR
There were no significant differences in grip strength measurements, auditory response, static righting reflex and pupillary reflex between the control group and treatment groups. In the females of the mid-dose group, the number of motor activity measurements (high sensor) was significantly increased, mainly due to one very active rat (control: 1285 ± 312, 150 mg/kg bw/day: 3315 ± 1313). As no similar effect was observed in the measurements by the low sensor, in the female high-dose group (absence of dose-related trend), or in the male treatment groups, this is considered to be an incidental occurrence.

ORGAN WEIGHTS
The absolute and relative organ weights of the treatment groups were not significantly different from the control group.

GROSS PATHOLOGY
1/5 males in the mid-dose group had exophthalmus of the right eye and 1/5 females in the control group had fluid in the uterus. These are both occasional findings in rats of this strain and age and are not treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC
3/5 males and 2/5 females in the control group had minimal to mild plasmacytic hyperplasia of the mandibular lymph nodes, which was also observed in 5/5 males and 2/5 females in the high-dose group. As these are common changes in this tissue, this is not considered to be a treatment-related observation. The thymus atrophy noted in 2/5 females in the high-dose group is also a common observation and therefore not considered to be of toxicological significance. The single male with non-treatment related exophthalmus of the right eye had a haemorrhage in the same eye. No further microscopic findings were noted.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No substance-related effects were observed up to and including the highest dose level

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion