Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 5th, 2009 - April 24th, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

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 407 (Repeated Dose 28-Day Oral Toxicity 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): SH-1
- Physical state: White powder
- Analytical purity: > 99%
- Batch No.: 20090213
- Expiration date of the lot/batch: 13 February 2011
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: males: about 220 g, females: about 160 g
- Fasting period before study: no data
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type)
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: March 13th, 2009 To: April 24th, 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility of the test substance
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours.
The analytical method used was validated under NOTOX project 490629.
Duration of treatment / exposure:
28 days
Duration of recovery: 14 days
Frequency of treatment:
once daily, 7d/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control:
not required

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to start of treatment and at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION
- weekly

WATER CONSUMPTION AND COMPOUND INTAKE:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery phase
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight for up to 20 hours
- How many animals:
- Haematological parameters checked: WBC, Diff leucocyte count, Red blood cells, Hb, Haematocrit, MCV, MCH, MCHC, platelets, clotting potential.
- Clinical biochemistry Parameters checked: ALAT, ALAT, ALP, total protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observations:
During week 4 of treatment, the following tests were performed on all animals:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0= normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerized monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Since the above mentioned measurements did not reveal treatment-related effects, the functional observation tests were not performed at the end of the recovery phase.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
The following organ weights and terminal body weight were recorded on the scheduled day of necropsy:
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart Uterus (including cervix), Kidneys, Prostate1, Liver, Seminal vesicles including coagulating glands (1), Ovaries, Thyroid including parathyroid (1)
(1 = weighed when fixed for at least 24 hours).
Statistics:
The following statistical methods were used to analyze the data:
- 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. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

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:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities and clinical signs of toxicity were noted during the observation period.
Salivation as seen after dosing among all dose groups (with the highest incidence at 1000 mg/kg/day) was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing, during treatment only). Incidental findings that were noted included chromodacryorrhoea, maculate erythema of the right ear, a dark right eye and opacity of the right eye. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant changes in body weights and body weight gain were noted.
Any statistically significant variations in body weights or body weight gain were of a slight nature and remained within the range considered normal for rats of this age and strain. These variations included lower body weights for females at 1000 mg/kg/day at the end of treatment, and lower body weight gain for females at 300 and 1000 mg/kg/day at the end of treatment and for females at 1000 mg/kg/day also on day 1 and 14 of the recovery period.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Any statistically significant changes at the end of the treatment period were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain. These changes included lower haemoglobin levels in males at 30 and 1000 mg/kg/day, lower relative eosinophil counts in females at 30, 300 and 1000 mg/kg/day, and lower platelet counts in females at 30 and 1000 mg/kg/day. The lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in females at 1000 mg/kg/day were only slightly outside the range considered normal for rats of this age and strain, but since these variations were minor and without a dose-related trend, they were considered to be without toxicological significance. Statistically significant changes at the end of the recovery period were also within the range considered normal for rats of this age and strain, and occurred in the absence of similar or more pronounced changes at the end of the treatment period. No toxicological significance was therefore ascribed to these changes, which included higher reticulocyte counts and lower mean corpuscular haemoglobin concentration (MCHC) in males at 1000 mg/kg/day, and higher red cell distribution width (RDW) and lower platelet counts in females at 1000 mg/kg/day.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters at the end of the treatment period distinguished treated animals from control animals:
− Lower cholesterol levels for females at 300 and 1000 mg/kg/day,
− Lower total bilirubin levels in males at 30, 300 and 1000 mg/kg/day,
− Lower bile acid levels in males at 300 and 1000 mg/kg/day.
These changes had recovered at the end of the recovery period. The lower total bilirubin and bile acid levels occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. At the end of the recovery period, creatinine and potassium levels were higher in females at 1000 mg/kg/day. These values exceeded the range considered normal for rats of this age and strain.
The statistically significant higher inorganic phosphate level in males at 1000 mg/kg/day at the end of the recovery period was absent at the end of the treatment period and remained within the range considered normal for rats of this age and strain.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment.
The statistically significant lower motor activity counts of males at 30 and 300 mg/kg/day as recorded by the high sensors occurred in the absence of a dose related trend. Therefore, these changes were considered to be without toxicological relevance.

ORGAN WEIGHTS
Liver weight and liver to body weight ratios of males at 300 and 1000 mg/kg/day, and liver to body weight ratios of females at 1000 mg/kg/day were slightly higher at the end of the treatment period, achieving a level of statistical significance for liver to body weight ratios only.
Thyroid weight and thyroid to body weight ratio was slightly higher for females at the end of the treatment period.
At the end of the recovery period, (relative) liver and thyroid weights were similar to control levels.
Other statistically significant changes at the end of the treatment period were considered to be of no toxicological significance as these were within the range considered normal for rats of this age and strain and/or occurred without a dose-related trend. These variations included higher relative seminal vesicle weight for males at 1000 mg/kg/day, higher relative testes weight for males at 30 mg/kg/day, and lower thymus weight for females at 300 mg/kg/day. The statistically significant higher brain to body weight ratio of females at 1000 mg/kg/day at the end of the recovery period, was considered to be related to the slightly lower terminal body weight, and hence without toxicological relevance.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.
Reduced size of the seminal vesicles, fluid in the uterus, red discolouration of the clitoral glands and tan foci on the clitoral glands occurred within the range of background findings noted in this type of study, and considered to be of no toxicological significance. No macroscopic abnormalities were noted among males at 30 and 300 mg/kg/day and females at 30 mg/kg/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study a NOAEL for SH-1 of 1000 mg/kg/day was established.
Executive summary:

Wistar rats were treated according to OECD guideline 407 with SH-1 for 28 consecutive days by daily oral gavage at dose levels of 0, 30, 300 and 1000 mg/kg/day, followed by a 14-day treatment-free recovery period.

The test substance was formulated in polyethylene glycol 400. One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

No toxicologically significant changes were noted in any of the observed/measured parameters.