Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, no deviations from guideline

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

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: Approximately 6 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: Group housing of 5 animals per sex in Makrolon cages with sterilized saw dust as bedding material and paper as cage enrichment
- Diet: ad libitum; pelleted rodent diet (SM R/M-Z from SSNIFF Spezialitäten GmbH, Soest, Germany
- Water: ad libitum
- Acclimation period: At least 5 days before the start of treatment under laboratory treatment.
- Health inspection: Prior to commencementof treatment to ensure that the animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial flourescent light/12 hours darkness per day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were prepared daily within 6 hours prior to dosing.
- Mixing appropriate amounts with (Type of food): pelleted rodent diet SM R/M-Z from SSNIFF Spezialitäten GmbH, Soest, Germany
- Storage temperature of food: Ambient temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations at NOTOX and on information from the sponsor
- Concentration in vehicle: 6-60 mg/ml
- Amount of vehicle (if gavage): Dose volume: 5 ml/kg bw
- Purity: not stated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analysed on a single occasion during the in-life phase for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under protection from light was also determined (highest and lowest concentration). The analytical method used was validated under NOTOX project 488245.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100, 300 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 males, 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a 5 day range finding study (NOTOX project 490421)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: wekly

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: No

FOOD EFFICIENCY:
- Body weight gain data: Yes


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Once, immediately prior to scheduled post mortem examination at the end of treatment.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight; water was available
- How many animals: all animals.
- Parameters listed in the field "Any other information on material and methods incl. tables" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Once, immediately prior to scheduled post mortem examination at the end of treatment.
- Animals fasted: Yes, overnight; water was available
- How many animals: all animals
- Parameters listed in the field "Any other information on material and methods incl. tables" were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all dose groups, all animals
- Battery of functions tested: Hearing ability, pupillary reflex, stating righting reflex, grip strength, motor activity

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:
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No clinical signs of toxicity were noted during the observation period.
One control male, one male and four females at 30 mg/kg/day showed salivation (in a dose dependent incident), which was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and the minor severity of the effect and its time of occurence (i.e. after dosing).
One male animal at 300 mg/kg/day showed scabs on the right shoulder. This finding 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 , this was considered a sign of no toxicological significance.
No mortalty occurred during the study.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant changes in body weights and body weight gain were noted.
At the end of treatment, body weights and body weight gain of males at 300 mg/kgbw/day were slightly lower than those of control males, but remained within the range considered normal for rats of this age and strain.. Therefore, these findings were considerd of no toxicological significance.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals. No toxicologically significant changes in food consumption before or after allowance for body weight were noted.

OPHTHALMOSCOPIC EXAMINATION
Not performed

HAEMATOLOGY
There were no differences noted in haematological parameters between control and treated rats that were considered related to treatment with the substance
Changes in haematological parameters of one female at 100 mg/kg/day (no. 31) were considered of no toxicological significance as they occurred in the absence of a treatment-related distribution. These changes included lower red blood cell counts, higher relative reticulocyte counts, a higher red blood cell distribution width (RDW), lower haemoglobin levels and a lower haematocrit value.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Lower total bilirubin levels in males and females at 300 mg/kg/day.
- Higher cholesterol levels in females at 300 mg/kg/day.
Lower levels of bile acids in males at 100 and 300 mg/kg/day were considered to have arisen as a result of high control values. in the absence of corroborative findings in females, these findings were considered to be of no toxicological significance.

URINALYSIS
Not performed

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strenth were normal in all animals. The variation in motor activity did not indicate a relation with treatment.

ORGAN WEIGHTS
Higher liver weights and liver to body weight ratios were observed for males at 100 and 300 mg/kg/day and females at 300 mg/kg/day. Liver weights of these animals were only marginally outside the range considered normal for rats of this age and strain and occurred in the absence of supportive microscopic findings. Therefore, these findings were considered of no toxicological significance.
Higher thyroid weights in males at 100 mg/kg/day achieved statistical significance, but were considered of no toxicological significance as these were well within the range considered normal for rats of this age and strain and were only marginally different than thyroid weights of control males. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.
incidental findings among females of all groups included fluid in the uterus, reduced size of the left lateral and medial lobes of the liver, isolated reddish foci on the glandular mucosa of the stomach, dark red foci on the thymus, tan discolouration of the clitoral glands and a yellowish hard nodule on the left mandibular lymph node. One control male showed a reduced size of the left testis and epididymis. These findings are occasionally seen among rats used in these types
of studies and in the absence of a treatment-related distribution, they were considered of no toxicological significance. No macroscopic abnormalities were noted among males at 30, 100 and 300 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 with 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:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Some minor gross pathological effects and some biochemical parameters which were marginally outside the range considered normal for rats of this age and strain were observed. From the results observed in the study a NOAEL for the substance of 300 mg/kg bw/day was established.
Executive summary:

In a Repeated Dose, 28-day Oral Toxicity Study performed according to OECD guideline 407, 2008 the substance (in polyethylene glycol 400) was administered to groups of 30 Wistarrats male and female by oral gavage at dose levels of 30, 100 and 300 mg/kg bw/day for 28 days. The control group received the vehicle only.

All animals survived to study termination.

Higher liver weights, lower total bilirubin levels and/or higher cholesterol levels among males at 100 mg/kg bw/day and animals at 300 mg/kg bw/day were of a minor nature and values of these parameters were within or only marginally outside the range considered normal for rats of this age and strain. Furthermore, no supportive microscopic findings were recorded. Therefore these findings were considered of no toxicological significance.

No toxicological significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consiumption, haematology investigations, macroscopic examination and microscopic examination). An NOAEL of 300 mg/kg bw/day was established in this study