Registration Dossier

Administrative data

Description of key information

The no-observed-effect-level (NOEL) of FAT 60'253/A in rats was found to be 1000 mg/kg body weight/day

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:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Hanlbm:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:RCC Ltd Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf / Switzerland
- Age at study initiation:6 weeks
- Weight at study initiation:Males: 142 -187 grams (mean 161 grams) ; Females: 117 -159 grams (mean 143 grams)
- Housing:In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet (e.g. ad libitum):Pelleted standard Provimi Kliba 3433 (batch no. 02/00) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
- Water (e.g. ad libitum):Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period:Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3
- Humidity (%):40-70
- Air changes (per hr):10-15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours fluorescent light/12 hours dark (light period between 06.00 and 18.00), music during the light period.

Route of administration:
oral: gavage
Vehicle:
other: PEG 300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly.
FAT 60'253/A was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Lot/batch no. (if required):
a) 405374/1 10200
b) 405371/1 31700
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the Sponsor.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 50, 200 ad 1000 mg/Kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
30 males and 30 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:Based upon the results of a non-GLP 5-day doserange-finding study (RCC Study Number 777262) in which FAT 60'253/A was administered by gavage to 2 rats per group and sex. Animals showed no overt signs of toxicity.
- Rationale for animal assignment (if not random):Recognized by the international guidelines as the recommended test system.
Positive control:
No data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1 -3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during pretest, treatment and recovery and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
The following anticoagulants were used during blood collection:
Hematology: EDTA-K2
Methemoglobin: Lithium heparin 30 I.U./ml
Coagulation: Sodium citrate, 3.8% (1 part anticoagulant to 9 parts blood)

CLINICAL CHEMISTRY: Yes
The following anticoagulant was used during blood collection:
Clinical biochemistry: Lithium heparin 15 I.U./ml;
The following commercial reference controls were used to monitor the performance of the method:
Clinical Biochemistry: Precinorm U (normal range); Precipath U (abnormal range) (Roche Diagnostic GmbH, Mannheim/Germany)
Lyphochek, Level 1 (normal range); Lyphochek, Level 2 (abnormal range)
(Bio-Rad Laboratories, ECS Division, Anaheim, California/USA)

URINALYSIS: Yes

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Slides of all organs and tissues listed in boldface type which were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist.
Other examinations:
None
Statistics:
The following statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all ratios, as well as clinical laboratory data :
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Student's T-Test was applied to locomotor activity and grip strength.
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
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:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until scheduled necropsy.
There were no findings noted during the weekly observations (weeks 1-3).

BODY WEIGHT AND WEIGHT GAIN
The mean body weights and the mean body weight gain of the test item treated animals were similar to those of the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The mean daily food consumption and the relative food consumption of the test item treated groups compared favorably with those of the control animals.

HAEMATOLOGY
After the four-week treatment period, the activated partial thromboplastin times of test itemtreated males were slightly prolonged when compared with the control males. Although the differences noted in males treated with 50 mg/kg/day and 1000 mg/kg/day attained statistical significance (p<0.05), they remained within the 95% confidence limits of the historical control data and were therefore considered to be incidental.
After the two-week recovery period, the ratio of low fluorescence reticulocytes was significantly lower (p<0.01) in males previously treated with 1000 mg/kg/day. This finding was considered to be of no toxicological relevance as similar differences were not evident in the females previously treated with 1000 mg/kg/day.
All other hematology parameters compared favorably after the treatment and recovery periods.

CLINICAL CHEMISTRY
Plasma urea levels were significantly higher (p<0.05) and creatinine levels were significantly lower (p<0.05) in males treated with 50 mg/kg/day, when compared with the controls. Males treated with 200 mg/kg/day or 1000 mg/kg/day were unaffected. The activity of gamma-glutamyltransferase was significantly less (p<0.01) in males treated with 50 mg/kg/day and 200 mg/kg/day when compared with the controls. These differences were considered to be incidental, as males treated with 1000 mg/kg/day were unaffected.
Minor differences noted in various clinical biochemistry parameters after two weeks' recovery were considered to be incidental changes unrelated to the treatment with the test item. All remaining clinical biochemistry parameters compared favorably with those of the controls after four weeks' treatment and two weeks' recovery.

URINALYSIS
There were no test item-related differences noted between control animals and treated animals after four weeks' treatment or two weeks' recovery.

NEUROBEHAVIOUR
There were no findings noted during functional observational battery (week 4).
Grip Strength
The mean hindlimb grip strength of males treated with 200 mg/kg/day or 1000 mg/kg/day was significantly (p<0.05) less than that of control animals. In the absence of similar results in females these findings were considered to be incidental.
Locomotor Activity
The mean locomotor activity was significantly reduced (p<0.05) in males treated with 50 mg/kg/day during the last measurement interval (45-60 minutes) and in males treated with 1000 mg/kg/day during the first measurement interval (0-15 minutes). Conversely, females treated with 1000 mg/kg/day were significantly more active (p<0.05) during the last measurement interval (45-60 minutes). These differences were considered to be incidental. The mean locomotor activity of the remaining groups compared favorably.

ORGAN WEIGHTS
After 4 Weeks
Although the mean absolute liver and kidney weights noted in males treated with 1000 mg/kg/day were significantly higher (p<0.05) than those of the controls, the relative liver and kidney weights of these males were unaffected. The absolute and relative organ weights of the test item-treated females compared favorably with those of the controls.
After 6 Weeks
The absolute and relative organ weights of the test item-treated animals were comparable with those of control animals.
Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified

None

Conclusions:
Based on the results of this study, 1000 mg/kg body weight/day of FAT 60'253/A was established as the no-observed-effect-level (NOEL).
Executive summary:

In this subacute toxicity study, FAT 60'253/A was administered daily by oral gavage to SPF bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Oral administration of FAT 60'253/A to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted in no changes of toxicological relevance in mortality, food consumption, body weight development, clinical signs (daily or weekly), functional observational battery (including grip strength and locomotor activity), parameters of hematology, clinical biochemistry or urinalysis, organ weights, macroscopic and microscopic findings.

Based on the results of this study, 1000 mg/kg body weight/day of FAT 60'253/A was established as the no-observed-effect-level (NOEL).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat

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

Mode of Action Analysis / Human Relevance Framework

Additional information

A key was performed to study the subacute toxicity study of FAT 60'253/A. FAT 60253/A was administered daily by oral gavage to SPF bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Oral administration of FAT 60'253/A to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted in no changes of toxicological relevance in mortality, food consumption, body weight development, clinical signs (daily or weekly), functional observational battery (including grip strength and locomotor activity), parameters of hematology, clinical biochemistry or urinalysis, organ weights, macroscopic and microscopic findings.

Based on the results of this study, 1000 mg/kg body weight/day of FAT 60'253/A was established as the no-observed-effect-level (NOEL).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP guideline study

Justification for classification or non-classification

The no-observed-effect-level (NOEL) of FAT 60'253/A in rats was found to be 1000 mg/kg body weight/day