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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:
12 February 2007 to 11 April 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Days 1 and 2 animls of group 4 dosed 40 mg/kg/day instead of 100 mg/kg/day.Max time btwn form and dose of test substance >4 hours by 50 minutes on Day 3,stability in vehicle indicated to be > 96 hours.Temp lower than min.No dev. adversely affected results
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): MTDID 6675
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Analytical purity: Formulation tested is 30.0% active ingredient in water
- Purity test date: 12/20/2005
- Lot/batch no.: 140499-8/25
- Expiration date of the lot/batch:02 July 2008
- Storage condition of test material: Refrigerated (2-8 degrees C) in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 7 weeks.
- Weight at study initiation: Males (Means of Groups 1-4): 253-256 g, Females (Means of Groups 1-4): 181-190 g.
- Fasting period before study:
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mili Ltd), Surry, United Kingdom). No cage-enrichment was provided during overnight activity monitoring. Certificates of analysis are examined and then retained in the NOTOX archives.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany) ad libitum.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: At least 5 days before the start of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.9 to 22.6 degrees Celsius
- Humidity (%): 36-86%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 14 March 2007 To: 11 April 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: Groups 1-4 (0, 10, 30, 100 mg test article/kg/day respectively)
- Amount of vehicle (if gavage): Total dose is 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method used was validated under NOTOX project 481578.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week, with a maximum of 4 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were based on the results of an 8-day range finding study.
- Rationale for animal assignment (if not random): Random
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before treatment and weekly thereafter.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily
BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22, and 28.
FOOD CONSUMPTION AND COMPOUND INTAKE):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Collected at end of treatment before necropsy.
- Anaesthetic used for blood collection: Yes, under iso-flurane anaesthesia (Abbott Laboratories LTd. Zwolle, The Netherlands)
- Animals fasted: Yes
- How many animals: All animals.
- Parameters checked in table: WBC, differntial leucocyte count, RBC, reticulocytes, RBC distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular heamoglobin concentration, platelets, prothrombin time, and activated partial thromboplastin time were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Collected at end of treatment before necropsy.
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table alanine aminotransfterase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, and inorganic phosphate were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: All dose groups were examined
- Battery of functions tested: Hearing, pupilary reflex, static righting reflex and grip strength.
Sacrifice and pathology:
GROSS PATHOLOGY
HISTOPATHOLOGY

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted.
Mortality:
no mortality observed
Description (incidence):
No mortality occured during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
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):
Slightly decreased haemoglobin and haematocrit level was noted in males at 100 mg/kg/day. Minor statistically significant increase of activated partial thromboplastin time (APTT) noted in females at 30 mg/kg/day were considered to have arisen by chance. No dose response distribution was noted and all values were within the physiological range. Therefore, this finding was considered not to represent a change of biological significance. No changes in haematological parameters were noted in males at 0, 10, and 30 mg/kg/day and females at 0, 10, 100 mg/kg/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Increased levels of alkaline phosphatase (ALP), urea and inorganic phosphate were noted in males at 100 mg/kg/day. Glucose and potassium levels were increased in males at 20 and 100 mg/kg/day. Decreased level of bilirubin was noted in all males at 10, 30, and 100 mg/kg/day. Slight increased creatinine level and decreased calcium levels were noted in females at 100 mg/kg/day. No changes in clinical biochemical parameters were noted in females at 10 and 30 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative liver weight was increased in males at 30 and 100 mg/kg/day. In addition relative liver weight was increased in males at 10 mg/kg/day. Slight increase in absolute and relative adrenal weight was noted in females at 100 mg/kg/day. This statistically significant change was slight and all values were within the physiological range. No clear dose response was noted. The finding was therefore considered to have no toxicological significance. Not changes in absolute organ weight were noted in males at 10 mg/kg/day. No changes in absolute and relative oragan weight were noted in females at 10 and 30 mg/kg/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At macroscopic examination the liver was found to be enlarged in two males at 100 mg/kg/day. No macroscopic abnormalities were noted among males at 10 and 30 mg/kg/day and control females.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Diffuse midzonal/centrilobular hyperthrophy of hepatocytes was noted at minor degrees in all males at 10 and 30 mg/kg/day and at slight or moderate degree in all males at 100 mg/kg/day. Diffuse follicular hypertrophy/hyperplasia in thyroid glands was seen at increased incidence and severity up to slight in males at 100 mg/kg/day. All other 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.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic histopathological findings were noted.

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
The No Observed Adverse Effect Level (NOAEL) for the test article in this study was 10 mg/kg for males and 100 mg/kg for females.
Effect level:
ca. 10 - ca. 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for the test article in this study was 10 mg/kg for males and 100 mg/kg for females.
Executive summary:

The potential toxicity of the test article (liquid, supplied at 29.9 ± 0.1% in water, lot 140499-19/10) was evaluated in male and female Wistar rats when administered by daily oral gavage for 28 days following OECD Guideline No. 407. The test material was diluted in Milli-U water to the appropriate concentrations and stock solution strength and specific gravity were included in calculations. The test article was administered by oral gavage to 5 animals/sex/group at 0 (Milli-U water), 10, 30 and 100 mg/kg daily for 28 days. 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. There were no test material related findings for any dose group in clinical appearance, functional observations, body weight and food consumption. The following changes in serum levels were noted in males treated at 100 mg/kg: increased alkaline phosphatase, increased glucose (also in males at 30 mg/kg), increased urea, increased inorganic phosphate, increased potassium (also in males at 30 mg/kg), decreased hemoglobin, and decreased hematocrit. Findings in females were limited to increased creatinine and decreased calcium in the 100 mg/kg dose group. Liver weight was increased in males at 30 and 100 mg/kg which was supported by an increased incidence and severity of hepatocellular hypertrophy/hyperplasia. Increased incidence and severity of thyroid follicular hypertrophy up to 100 mg/kg in males was considered an adaptive change to the increases in liver. The increase in liver weight was considered adverse. In conclusion, the No Observed Adverse Effect Level (NOAEL) for the test article in this study was 10 mg/kg for males and 100 mg/kg for females.