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Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 18, 2007 to December 12, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed in accordance with GLP standards and with testing guidelines. No major deviations from the protocol. Minor deviations did not affect the integrity or outcome of the study.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
minor deviations
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
minor deviations
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: recognised supplier
- Age at study initiation: The preliminary study animals: 34 to 36 days old; The main study animals: 48 to 50 days old.
- Weight at study initiation: The preliminary study animals: 106.0 to 138.0 g (males), 113.0 to 124.1 g (females); The main study animals: 179.5 to 236.5 g (males), 140.4 to 173.2 g (females).
- Fasting period before study: no data
- Housing: single exclusive room during each set of assessments, otherwise the main study animals were housed in groups of five, and the preliminary study animals were housed in groups of three.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: The preliminary study animals: 7 days; The main study animals: 21 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 40 to 70% (on a few occasion the relative humidity was outside the ranges, highest recording was 80%)
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (0600h to 1800h) and 12 hours dark

IN-LIFE DATES: No data
Route of administration:
oral: gavage
Vehicle:
other: Purified water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily and used within 4 hours of preparation.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: refrigerated

VEHICLE
- Amount of vehicle (if gavage): a dose volume of 10mL/kg was used. Individual dose volumes were based on individual bodyweight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed weekly on the main study. The normal target range for the results was 90% to 110% (liquid formulations) of nominal concentration.
Duration of treatment / exposure:
Preliminary study, Test duration: 7 days
Main study, Test duration: 28 days
Frequency of treatment:
The preliminary study animals were dosed once daily for seven days, excluding the day of termination.
The main study animals were dosed once daily for 28 days excluding the day of necropsy.
Remarks:
Doses / Concentrations:
250, 500 and 1000 mg/kg/day (prelimary study)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 250, 500 and 1000 mg/kg/day (main study)
Basis:
actual ingested
No. of animals per sex per dose:
Preliminary study: 3 males and 3 females per dose
Main study: 5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

For the preliminary study:
The limit dose of 1000 mg/kg/day was selected as the high dose level for the preliminary study based on the results of the acute rat oral toxicity study (Covance study number 0665/349). In this study the acute median lethal oral dose was found to exceed 2000 mg/kg. For the preliminary study, a descending sequence of dose levels (250 and 500 mg/kg/day) were selected to aid in the selection of dose levels for the main study.

For the main study:
In the preliminary study, dose levels of 250, 500 or 1000 mg/kg/day were well tolerated following 7 days of repeated administration. The dose level of 1000 mg/kg/day was chosen as the high dose level for the main study because this is the maximum dose level (limit dose) required by the regulators. A descending
sequence of dose levels (250 and 500 mg/kg/day) were selected to further characterise the toxicity of the test article and/or to establish a No-Observable-Effect-Level (NOEL) and/or No-Observable-Adverse-Effect-Level (NOAEL).

- Rationale for animal assignment (if not random): randomisation procedure
- Rationale for selecting satellite groups: NA
- Post-exposure recovery period in satellite groups: NA
- Section schedule rationale (if not random): NA
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All main study and preliminary animals were observed daily for signs of ill health or overt toxicity after dosing (0.5, 1, 2 and 4 hours).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, each animal was given a detailed physical examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Day-7, the first day of the dosing, weekly intervals and on the day of necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g /animal/day (for the preliminary study) and g/animal/week (for the main study).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: NA

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: NA
- Dose groups that were examined: NA

HAEMATOLOGY: Yes
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection:No data
- Animals fasted: No data
- How many animals: 4 males, 4 females
- Parameters examined: hæmoglobin concentration, packed cell volume, mean cell volume, mean cell hæmoglobin concentration, red cell distribution width, platelet crit, platelet distribution width, red blood cell count, reticulocytes, mean cell hæmoglobin, hæmoglobin distribution width, platelet count, mean platelet volume, total and differential white cell count, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals:
- Parameters examined: aspartate aminotransferase, alkaline phosphatase, potassium, inorganic phosphorus, total protein, globulin, total cholesterol, urea, creatinine, alanine aminotransferase, sodium, calcium, chloride, albumin, albumin/globulin ratio, glucose, total bilirubin.

URINALYSIS: No
- Time schedule for collection of urine: NA
- Metabolism cages used for collection of urine: NA
- Animals fasted: NA
- Parameters examined: NA

NEUROBEHAVIOURAL EXAMINATION: Yes, a battery of behavioral tests was performed (FOB). Observational measurements were evaluated before and upon removal from the home cage. The animal was also placed into a circular arena for 2 minutes. During this time, behavior was evaluated as indicated in the Open Field section.
- Time schedule for examinations: Week 4 (end of study)
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity to stimuli, grip strength and motor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Gross lesions from all main study animals and tissues from control and high dose animals were examined microscopically by the study pathologist. Due to treatment related effects in the kidneys of females receiving 1000mg/kg/day the kidneys of females receiving 250 or 500 mg/kg/day were examined by microscopy by the study pathologist.

HISTOPATHOLOGY: Yes. Gross lesions from all main study animals and certain tissues from control and high dose animals were embedded in paraffin was BP, sectioned at a nominal 5 µm and stained with hæmatoxylin and eosin. Liver, spleen and kidney from low and intermediate dose animals were processed to the slide stage.
Statistics:
ANOVA, Levene's test
Clinical signs:
no effects observed
Description (incidence and severity):
no unsheduled deaths in the preliminary and main study
Mortality:
no mortality observed
Description (incidence):
no unsheduled deaths in the preliminary and main study
Body weight and weight changes:
no effects observed
Description (incidence and severity):
absence of any dose relationship
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
similar to control
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):
A slightly higher mean activated partial thromboplastin time (APTT) in both sexes at 1000 mg/kg/day. A slightly higher mean prothrombin time for males at 1000 mg/kg/day. All treated female groups , higher WBC count.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
The mean hindlimb grip strength was lower than control for males receiving 500 or 1000 mg/kg/day and higher than the control for females receiving 1000 mg/kg day.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
higher kidney weight for both sexes at 1000 mg/kg/day
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
very minor corticomedullary mineralisation in the kidney of all treated female groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths in the preliminary or main study. All clinical signs observed in the preliminary or main study were considered typical of laboratory maintained rats.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment related effects on Week 1-4 mean body weight gains in all treated groups. Mean body weight gains for males receiving 250 mg/kg/day and both sexes receiving 500 mg/kg/day were lower than contols. But this was considered to be of no relationship to treatment as the females receiving 250 mg/kg/day and both sexes receiving 1000 mg/kg/day were similar to the control.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Mean food consumption (Week 1-4) for all treated groups were similar to the control.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
Haematology investigations on Day 29 revealed a slightly higher than control mean activated partial thromboplastin time (APTT) in both sexes receiving 1000 mg/kg/day. The mean prothrombin time for males receiving 1000 mg/kg/day was slightly higher than control. All treated female groups were noted with a slightly higher than control mean lymphocyte count (and concomitantly higher mean WBC count), with a dose related trend evident. All other differences from control were either generally within the concurrent control range and/or showed no dose- relationship and were therefore considered to be unrelated to treatment.

CLINICAL CHEMISTRY
All differences from control were either generally within the concurrent control range, were attributable to one outlying value and/or showed no dose-relationship and were therefore considered to be unrelated to treatment.

URINALYSIS
Not examined

NEUROBEHAVIOUR
The mean hindlimb grip strength was lower than control for males receiving 500 or 1000 mg/kg/day and higher than the control for females receiving 1000 mg/kg/day. But these contradicting differences suggest an unlikely relationship to treatment. In the remaining tests from the functional observation battery or in the assesment of locomotor activity, clinical signs or histopathology of neurological or musculoskeletal tissues no treatment related effects were noted.

ORGAN WEIGHTS
The group mean, absolute kidney weight was higher than control for males and females treated 1000 mg/kg/day. These findings were considered to be of unlikely relationship to treatment.

GROSS PATHOLOGY
Most tissues were macroscopically unremarkable and the findings seen were generally consistent with the usual patterns og findings in animals of this strain and age. There were no macroscopic findings due to effects of the test article.

HISTOPATHOLOGY: NON-NEOPLASTIC
There was a dose-related increase in very minor corticomedullary mineralisation in the kidney of treated females. Corticomedullary mineralisation was characterised by small deposits of basophilic, granular material in the region of the outer stripe of the renal medulla.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No data

HISTORICAL CONTROL DATA (if applicable)
No data
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: original NCD unit is mg/kg/day.
Critical effects observed:
not specified
Conclusions:
The repeated oral dose toxicity of L-TEE was tested in a repeated oral gavage study in rats for 28 days. The study was performed in compliance with the regulatory guidelines OECD (407) and EC (Method B7). No adverse findings were noted in this study and the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

L-TEE was tested in a repeated oral gavage study in rats. L-TEE was administered at doses 250, 500 or 1000 mg/kg/day to rats for 28 days. The study was performed in compliance with the regulatory guidelines OECD (407) and EC (Method B7).

The No-Observed-Effect-Level (NOEL) could not be established in this study because of the minimal/slight corticomedullary mineralisation in the kidneys and increased lymphocyte counts in females treated at 250, 500 or 1000 mg/kg/day. However, no adverse findings were noted in this study and the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was performed in accordance with GLP standards and with test guidelines. No major deviations from the protocol. Minor deviations did not affect the integrity or outcome of the study.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The repeated oral dose toxicity of L-TEE was tested in a repeated oral gavage study in rats for 28 days. The study was performed in compliance with the regulatory guidelines OECD (407) and EC (Method B7). No adverse findings were noted in this study and the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 1000 mg/kg/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This is a key study.

Justification for classification or non-classification

L-TEE was shown to have a low oral repeated-dose toxicity hazard when tested in a 28-day toxicity study and the identified NOAEL was 1000 mg/kg/day.

Based on this result, L-TEE is not hazardous and is not classified according to CLP or DSD.