Pentyl 2-cyano-(3-(6-methoxybenzothiazol-2-ylimino)-2,3-dihydro-1H-isoindol-1-ylidene)acetate

Endpoint summary

Description of key information

A NOAEL and NOEL of 1000 mg/kg bw/day was derived based on no treatment-related effects observed at any of the dose levels tested in the 28-d oral study in rats.

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:

From Aug. 19, 2000 to Mar. 08, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat has proved to be a suitable species for subacute oral toxicity testing with many different substances and is the species of choice according to the international guidelines

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HARLAN WINKELMANN Gartenstr. 27, D-33178 Borchen, SPF breeding colony
- Age at study initiation: Approximately 6 wk
- Housing: Macrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet: ssniff R/M (V1534), ad libitum, except for the period in which the animals were kept in diuresis cages
- Water: Tap water in plastic bottles, ad libitum, except for the period in which the animals were kept In diuresis cages
- Acclimation period: At least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C
- Humidity (%): 50±20 °C
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

IN-LIFE DATES: From Sep. 19, 2000 to Oct. 318, 2000

Route of administration:

oral: gavage

Details on route of administration:

The oral route is considered to be a potential exposure route in man.

Vehicle:

other: Tylose H 4000 G4 PHA, (0.5 % in deion. water)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance was suspended in the concentrations of 1.25, 5.0 and 20 % in Tylose H 4000 G4 PHA (0.5% in deionized water). After each measurement of the body weight, the calculation of the application volume was repeated. The final dosing volume was 5 mL/kg bw

VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance is insoluble in water but gives a stabe and homogeneous suspension in Tylose H 4000 G4 PHA (0.5% in deionized water)
- Concentration in vehicle: 1.25, 5.0 and 20 %
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Verification of concentration in formulation by HPLC in first and last week of treatment

Duration of treatment / exposure:

29 d, 28 applications

Frequency of treatment:

Once a day for 28 d

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5/sex/dose in main groups
5/sex/dose in recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment (if not random): Randomization using computer-generated algorithm
- Rationale for selecting satellite groups: To check the reversibility of the effects, if any
- Post-exposure recovery period in satellite groups: Yes

- Rationale for dose-selection: Acute oral toxicity testing of FC 84508 at a dose of 2000 mg/kg in the rat (only this dose was tested) showed that the median lethal dose (LD50) is above 2000 mg/kg body weight in both male and female animals. The dose of 2000 mg/kg was tolerated by all the animals without signs of intoxication.
Based on these results, dose levels of 62.5, 250 and 1000 mg/kg body weight per day were selected.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily in all groups

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before start of the study and once a week during the study

BODY WEIGHT: Yes
- Time schedule for examinations: weekly once

FOOD CONSUMPTION:
- 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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: Weekly once

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes (intraperitoneal injection of 67 + 6.7 mg/kg bw Ketamine HCl + Xylazine)
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes, killed by section of the vena cava cranialis in deep narcosis (intraperitoneal injection of 67 + 6.7 mg/kg bw Ketamine HCl + Xylazine) and exsanguinated

URINALYSIS: Yes
- Time schedule for collection of urine: Few days before termination of the study as well as before the end of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before start of the study and once a week during the study
- Dose groups that were examined: All groups
- Battery of functions tested: sensory reactivity, grip strength and motor activity

OTHER: Animals were also examined for opacity of the refracting media of the eyes, damage to the oral mucosa and impairment of dental growth

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were necropsied and checked for macroscopically visible abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

HISTOPATHOLOGY: Yes, Following tissues/organs were preserved in a suitable fixative and processed for histopathological investigations: adrenal gland, bone marrow (sternum), brain, heart, colon, jejunum, kidneys, liver, lungs, lymph nodes (mandibular and iliac), ovaries, uterus, thyroid and parathyroid glands, prostate gland, spleen, stomach, testis, epididymides, thymus, trachea, urinary bladder, N. ischiadicus and spinal cord(cervical),

Other examinations:

Following organs were weighed and the organ to body weight ratios calculated:
Heart , liver, kidneys, adrenal glands, spleen, testes, epididymides, thymus and brain

Statistics:

Evaluation was performed by l/S DI&A, Aventis Pharma Deutschland GmbH, with the aid of a program package for the evaluation of toxicological studies.

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 examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

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

Details on results:

CLINICAL SIGNS AND MORTALITY: No significant treatment related clinical signs were observed. No deaths occurred throughout the study.

BODY WEIGHT AND WEIGHT GAIN: Body weight gains were statistically significant increased in female animals from the high dose group from Day 25 to 29 of the study, which was an incidental finding

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption remained unaffected by the administration of the test substance throughout the study in all dose groups

WATER CONSUMPTION AND COMPOUND INTAKE: Administration of the test substance did not alter the water consumption of the rats

HAEMATOLOGY: A decrease in the coagulation time in females of the intermediate and high dose group was observed at the end of the 28-d treatment period, which was considered to be an incidental finding

CLINICAL CHEMISTRY: Main group: Higher values of chloride in males of the high dose group and of cholesterol in both sexes of the high dose group and decreases in triglyceride values in males in all dose groups were found.
Recovery group: Increases in sodium, calcium, cholesterol, triglycerides, total protein and albumin values as well as a decrease in glucose in males of the high dose group and decreases in uric acid and triglycerides in females of the high dose group were noted.
All these effects were considered incidental effects.

URINALYSIS: Urine volume was significantly decreased in female animals of the high dose group after the 28-d treatment period, which was considered to be an incidental effect

NEUROBEHAVIOUR: Neurotoxicological measurements including 'open field' observations, assessment, of sensory function, and forelimb and hindlimb grip strength were not influenced by the administration of the test substance in all groups.

ORGAN WEIGHTS: Remained unaffected by the administration of the test substance

GROSS PATHOLOGY: Relevant macroscopical changes could not be seen in male or female animals

HISTOPATHOLOGY: NON-NEOPLASTIC: Sporadic histopathological findings were observed in various organs of different animals, randomly distributed over all investigated groups. Type and incidence of these changes were not relevant for the assessment of the test compound

HISTORICAL CONTROL DATA (if applicable): Changes concerning clinical chemistry were within the historical control data range

OTHER FINDINGS: No opacity of the refracting media of the eyes, changes of the oral mucosa or impairment of dental growth was observed.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

Under the test conditions, the NOAEL and NOEL of the test substance were determined to be above 1000 mg/kg bw/day in rats

Executive summary:

A study was conducted to assess the sub acute repeated dose toxicity of the test substance in rats according EU Method B.7. and OECD guideline 407 in compliance with GLP

 

Groups of male and female rats received test substance by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day for a period of 28 d. On Day 29, 5 males and five females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 d.

 

Behavior and state of health were observed daily in all groups, Body weights and food consumption were recorded twice weekly, water consumption once weekly. Once before the first treatment and once a week thereafter detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa and impairment of dental growth.

 

Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hindlimb grip strength were conducted at the end of the treatment period. Hematological examinations, clinical chemistry and urine analysis were carried out at the end of the treatment period and after the recovery period.

 

During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, hematological and clinical chemistry data, urine data (volume, specific weight), absolute and relative organ weights and neurotoxicological measurements (motor activity, forelimb and hindlimb grip strength) were analyzed with the aid of a statistical program.

 

No deaths occurred throughout the study. Behavior, state of health, neurotoxicological, hematological and blood serum parameter analysis remained unaffected by the administration of the test substance in all dose groups.

 

Body weight gain, organ weights and food and water consumption were comparable in control and dose groups. No test substance related changes were detected by urine analysis or at necropsy or histopathology.

 

In conclusion, repeated administration of test substance did not cause any substance related changes at the doses administered. Hence, under the test conditions, NOAEL and NOEL of the test substance were determined to be above 1000 mg/kg bw/day in rats.

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

Additional information

A study was conducted to assess the sub acute repeated dose toxicity of the test substance in rats according EU Method B.7. and OECD guideline 407 in compliance with GLP

Groups of male and female rats received test substance by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day for a period of 28 d. On Day 29, 5 males and five females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 d. Behavior and state of health were observed daily in all groups, Body weights and food consumption were recorded twice weekly, water consumption once weekly. Once before the first treatment and once a week thereafter detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa and impairment of dental growth. Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hindlimb grip strength were conducted at the end of the treatment period. Hematological examinations, clinical chemistry and urine analysis were carried out at the end of the treatment period and after the recovery period. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Organs and tissues were processed for histopathological examination and checked for microscopically visible changes. Body weights, hematological and clinical chemistry data, urine data (volume, specific weight), absolute and relative organ weights and neurotoxicological measurements (motor activity, forelimb and hindlimb grip strength) were analyzed with the aid of a statistical program.

No deaths occurred throughout the study. Behavior, state of health, neurotoxicological, hematological and blood serum parameter analysis remained unaffected by the administration of the test substance in all dose groups. Body weight gain, organ weights and food and water consumption were comparable in control and dose groups. No test substance related changes were detected by urine analysis or at necropsy or histopathology. In conclusion, repeated administration of test substance did not cause any substance related changes at the doses administered. The NOAEL and NOEL of the test substance were determined to be > 1000 mg/kg bw/day in rats (Dr. Seeberger A and Dr. Kauffmann HM, 2001).

 

A NOAEL and NOEL of above 1000 mg/kg bw/day was derived based on no treatment-related effects observed at any of the dose levels tested in the 28-d oral study in rats.

Justification for classification or non-classification

A NOAEL and NOEL of above 1000 mg/kg bw/day was derived based on no treatment-related effects observed at any of the dose levels tested in the 28-d oral study in rats and therefore based on the results the test substance does not meet the requirement for classification according to according to EC criteria (67/548/EEC) and according to CLP criteria (EC 1272/2008).