Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 June 2005 - 20 October 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000. .
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Description: A clear colourless liquid
Storage conditions: Room temperature in the dark
Lot number: 52834
Expiry date: December 2006
Purity: >98%

Test animals

Species:
rat
Strain:
other: Crl:CD
Details on species / strain selection:
(SD)IGS BR rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 45 to 49 days
- Weight at study initiation: 221.3 to 257.3 g for males and 158.8 to 190.1 g for females.
- Fasting period before study: overnight before routine blood sampling
- Housing:The cages were made of a stainless steel body with a stainless steel mesh lid and floor
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): freely available
- Acclimation period: 12 days
- Environmental enrichment: chew block

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): passed to atmosphere and not re-circulated
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours continuous dark per 24 hours

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Animals received the test substance or vehicle control formulations orally using a suitably graduated syringe and a rubber catheter (Ch 8) inserted via the mouth into the stomach.
Vehicle:
water
Remarks:
for formulation
Details on oral exposure:
Method of administration:
Gavage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas liquid chromatograph (GLC)
A representative sample of test formulation was accurately weighed and dissolved in a suitable volume of acetone. The extract was diluted using acetone to provide a solution containing the test item at an expected concentration in the range 40 µg/ml - 80 µg/ml.
- Linearity was confirmed over the nominal concentration range 20 µg/ml – 100 µg/ml with a coefficient of determination >0.995.
- The precision of injection was <5% for six replicate injections of standard solutions containing the test ietm at nominal concentrations of 20 µg/ml and 100 µg/ml
- Mean procedural recovery value of 94.0% (CV=5.89%, n=6) was obtained for 1 mg/ml and 99.9% (CV=2.47%, n=6) was obtained for 100 mg/ml.
- limit of detection: 0.12 mg/ml
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
No. of animals per sex per dose:
Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 15 mg/kg bw/day
Male: 5 animals at 150 mg/kg bw/day
Male: 5 animals at 1000 mg/kg bw/day
Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 15 mg/kg bw/day
Female: 5 animals at 150 mg/kg bw/day
Female: 5 animals at 1000 mg/kg bw/day
Details on study design:
Administration
Observations: Clinical, Detailed physical examination and arena observations, Neurobehavioural screening, Sensory reactivity, Bodyweight, Motor activity, Food consumption, Haematology
Necropsy: after 4 weeks of treatment

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day -7, Day 1, weekly throughout the treatment, Day 28, before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 of treatment
- Anaesthetic used for blood collection: Yes general anaesthesia
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in 'List of parameters checked for Haematology' were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 of treatment
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in 'List of parameters checked for Clinical chemistry' were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 4 of treatmen
- Dose groups that were examined: each group
- Battery of functions tested: sensory activity / grip strength / motor activity / Touch response / Auditory startle response / Tail pinch response
Sacrifice and pathology:
GROSS PATHOLOGY: Yes:
Adrenals Liver
Brain Ovaries
Epididymides (Epididymid) Spleen
Heart Testes
Kidneys Thymus

HISTOPATHOLOGY: Yes
Statistics:
All statistical analyses were carried out separately for males and females.
All analyses were carried out using the individual animal as the basic experimental unit.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs were observed throughout the study.
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths were observed throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall group mean bodyweight gains for all treated groups were similar to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption values for all treated groups were similar to control throughout the study
Food efficiency:
no effects observed
Description (incidence and severity):
Overall group mean food conversion efficiency values for all treated groups were similar to controls.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean APTT values were statistically significantly lower than controls for all treated groups, however as the individual values for each group were within the expected background range for animals of this age and strain (APTT rat: ♂ 1 percentile 11.6, mean
19.76, 99 percentile 28.5, ♀ 1 percentile 8.5, mean 16.49, 99 percentile 24.3), there was no dose relationship for the females and no treatment-related histopathology findings, this finding is considered not to be of toxicological importance.
There were no other differences from controls, which were considered possibly associated with treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A higher than control group mean albumin value was recorded for females receiving 1000 mg/kg/day accompanied by a higher than control A/G ratio. These effects of treatment were not supported by any histopathological or other corroborative findings from the study and thus were considered not to be of toxicological importance.
A higher than control group mean sodium value was recorded for females receiving 150 mg/kg/day, however as the sodium values for females treated at 1000 mg/kg/day were comparable with controls, this finding is considered not to be treatment-related.
There were no other differences from controls, which were considered possibly associated with treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute adrenal and thymus weights were higher than controls for males receiving 1000 mg/kg/day, however there were no corresponding macroscopic or microscopic changes. Bodyweight adjusted kidney weights were statistically significantly higher than controls for
females receiving 150 or 1000 mg/kg/day, however as no dosage relationship was observed and there were no corresponding macroscopic or microscopic findings this finding is considered not to be treatment-related.
There were no other differences from controls, which were considered possibly associated with treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed at termination revealed no changes attributable to treatment with the test item.
Testes:
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
Clinical observations:
No unscheduled deaths, treatment related clinical or neurobehavioral signs were observed throughout the study.
Overall group mean bodyweight gains, food consumption values and food conversion efficiency for all treated groups were similar to controls.

Laboratory findings:
Group mean APTT values were lower than controls (Range:M:15- 18% + F:12-23%) for all teated groups. However, as the individual values were within the background data range,there was no dose-relationship for the females and no
treatment-related histopathology findings, this finding is considered not to be of toxicological importance.


A higher than control mean albumin value was recorded for females receiving 1000 mg/kg/day accompanied by a higher than control A/G ratio. These effects of treatment were not supported by any histopathological or other corroborative findings from the study and thus were not considered to be of toxicological importance.

Effects in organs:
Absolute adrenal and thymus weights were higher than controls for males receiving 1000 mg/kg/day.


The macroscopic and microscopic examinations performed at termination revealed no changes in attributable to treatment.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
mortality
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
mortality
organ weights and organ / body weight ratios
Remarks on result:
not determinable

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: all
Organ:
other: none

Applicant's summary and conclusion

Conclusions:
It is concluded that oral administration of MBD to rats for four weeks did not result in any findings of toxicological importance. Therefore 1000 mg/kg/day is regarded as the highest No Observed Adverse Effect Level (NOAEL) on this study.