N-(cyclohexylthio)phthalimide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(cyclohexylthio)phthalimide

EC Number:

241-774-1

EC Name:

N-(cyclohexylthio)phthalimide

Cas Number:

17796-82-6

Molecular formula:

C14H15NO2S

IUPAC Name:

N-(cyclohexylthio)phthalimide

Details on test material:

Lot No.: 041/07
Purity: 99.33%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Velaz Praha, Czech republic
- Age at study initiation: no data
- Weight at study initiation: mean initial body weight of animals was 195.99 g males, and 186.56 females
- Fasting period before study: no data
- Housing: cages (3 animals per cage) in the experimental animal house
- Diet (e.g. ad libitum): normal laboratory food (Top Dovo, PD Horné Dubové) in recommended dose
- Water (e.g. ad libitum): inlimited drink water
- Acclimation period: 6 days before study

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22±2°C
- Humidity (%): 40-70%
- Air changes (per hr): central air-condition
- Photoperiod (hrs dark / hrs light): 12/12 artificial

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Duslin is slightly solluble in water.
- Concentration in vehicle: 5-20-40 mg/kg
- Amount of vehicle (if gavage): 0.5ml/100g of body weight
- Lot/batch no. (if required): Lot No.: L 809182 from G.Heess, Germany
- Purity:no data

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The tesst substance was administrated per os with help of metal stomach tube every day for 90 days. Control and highest dose group were observed 28 days after last application.

Frequency of treatment:

substance administration - daily for 90 days
animals were weighted once in two days
During recovery period (28days) reversibility, persistence or delayed occurrences of eny effect were investigated. The clinical observation, body weight and food consumption were recorded.
Blood for hematological and clinical chemistry investigation was collected before application, after 60 days and 90 days of application of test substance.
The urine analysis was performed at the end of the study.
All recovery animals were sacrificed after the end of recovery period.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5 mg/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
20 mg/kg
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
40 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

total number: 78 males + 78 females
No. of animal per dose: 15 males + 15 females
No. of animal per control: 15 males + 15 females
No. of animal per satellite: 9 males + 9 females

Control animals:

yes

Details on study design:

- Dose selection rationale: the same dose as in 28 days study were used, The low dose level -5mg/kg was estimated from available information about substance, the highest dose level -40mg/kg- represents 8-fold of the low dose level.
- Post-exposure recovery period in satellite groups: 28 days

Examinations

Observations and examinations performed and frequency:

Clinical observation: were made once a day, 1 hour after application of the test article. The helth condition of the animal, reaction of the animals to the applied substance, their well being were monitored every day and recorded in protocols.

Body weight and Food consumption: individual weighting of the animal was performed every a two days. The body weight of the animals was recorded in Application notebooks. Food consumption was monitored once a week and recorded in protocols.

Sampling and Processing of the Biological Material: The sampling and processing of biological material were performed according to relevant SOP: blood collection for hematological investigations from the caudal incisium, the blood collection for measurement of Quick test from the heart, the blood collection for clinical chemistry from retrobulbar venoplex, the processing of the blood and determination of its hematological and clinical chemistry parameters. The sampling of blood was made before the first application of the test article (0 sample), after 60 days of the test article application (1st sample) , at the end of the application (2nd sample), in animals of the satellite groups 28 days after the last application of the test article (3rd sample).
The urine was collected at the end of the study. Timed urine volume collection has been used, the animals were placed into metabolic cages for 6 hours.

Hematology: The hematocrit, hemoglobin, leukocytes, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets using hematological analyser MICROS 60 were measured. A standard blood sample Minotrol 16 and aabs Pack LMGE (ABX) were used for calibration of the instrument and measurement of the sample. The differential count was estimated microscopically, blood smears were coloured according to Giems-Romanowski. After the end of test article application and in animals of satellite groups Quick test was used for blood clotthing time evaluation.

Clinical Chemistry: The alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, glucose, total cholesterol, triacylglycerols, creatinine, urea, total bilirubin, total proteins, albumin, calcium, inorganic phosphorus were analysed. The biochemical analyser Lisa 200 of the Hycel Co. was used for all measurements. For the measurements of natrium, kalium and chloride the electrolyte analyzer 9180 AVL of the AVL Co. was used. The quality control of the analysis was assured by the control serum Lyonorm and Precinorm-U.

Urine Anlysis: The specific gravity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood were identified using the test strips Deka PHAN and LAURA Smart, reflectance photometer. Timed collection of urine during 6 hours was used.

Sacrifice and pathology:

Pathology: After finishing the test the animal were anesthetized with diethyl ether, exsanguinated from the heart and necropsied. The autopsy was made in acordance with standard operation procedures.

Gross Necropsy: All animals were subjected to a full, detailed gross necropsy, which insludes careful examination of external surface of the body, all orifices and cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, heart, brain, spleen, thymus, seminal glands, uterus and ovaries of all animals were weighted. The weighted organs and lungs, lymph nodes, bone marrow, stomach, small and large intestines, trachea, thyroid, esophagus, aorta, salivary gland, urinary bladder, peripherial nerve, brain, pituitary, males and females reproductive organs of the control and high dose groups were processed for microscopic examination. All organs and tussues were fixed in formalin 1:9 and prepared in the paraffine technical. The tissue segments of rats organs were sectioned in 10um slices in Reichert Jung and Reichert. All histological slices were stained with hematoxylin and eosin. The segments of spleen were stained by Liesegang. The histological preparations were examined under light microscopes JENAMED 2, BAT-400-TH-5. Relative weights of organs were evaluated using analysis of variance (ANOVA).

Statistics:

Individual results obtained during the experiment were statistically evaluated by statistical programme Statgraphics. Statistical evaluation was made separately for males and females. To identify homogeneity groups the Bartllet test was used. In the case of homogeneity One-Way Analysis of variance with consecutive Multiple Ranges tests was acomplished. In case of non homogeneity Kruskal-Wallis One Way Analysis by Ranks was applied.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Three deaths occured. Reason of death was aspiration of the test substance after application . Deaths were clasified as accident.

Mortality:

no mortality observed

Description (incidence):

Three deaths occured. Reason of death was aspiration of the test substance after application . Deaths were clasified as accident.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of all animals moderately increased.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

All significant defferences in hematology parameters which were found can be considered to be without toxicological importance because no dose related relationship was evident. They can be explaining as the result of intraindividual and interindividual va

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

increase activity of ALP in males of all dose group and females of dose group 40 mg/kg

Urinalysis findings:

no effects observed

Gross pathological findings:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals lived through observation period without important signs of intoxication. There were three unscheduled deaths. Animal died after application the test article. Reason of these deaths was aspiration the test article and these death were classified as accident. During the application of Duslin occurrence of smooth stool was observed in animals of all dose groups. The smooth stool did not repeat in these animals, it was registered only once or two times. The dose dependence was not registered. Clinical observation did not reveal relevant changes of health or negative reaction in satellite animals.

BODY WEIGHT AND WEIGHT GAIN
was statistically analysed weekly (1-13 week). Additionally, body weight was statistically analysed in the satellite animals 1st-4th week post-treatment observation. During the study the body weight of all animals moderately increased. Significant differences between control group and dose groups were registered in some cases. Thi one did not show any treatment - related differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A normal laboratory diet was served in recommended doses. Intake of drinking water was unlimited. The food consumption in males and females of all dose groups was similar to the control group with exception of some cases. The link between food consumption and body weight in these cases was not registered.

FOOD EFFICIENCY

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY
The statistically significant changes ware registered in values of WBC and RBC in animals of all groups (including control). These changes have not direct connection with test article. The hemoglobin values of males control and all dose groups (except males of dose group of 5 mg/kg after 60 day of application of test article) were increased against starting values during all experiment. The values of hemoglobin in males of dose group 20 mg/kg after 90 day of application were increased against control group. In females control and dose groups of 5 and 40 mg/kg increased values of hemoglobin against starting values after 90 day application period were registered, too. The values of hemoglobin of satellite females dose group of 40 mg/kg were decreased against satellite control group.
Values of hematocrit in all participating males (except males dose group 5 mg/kg after 60 day application the test article) were increased against starting values during all experiments. Females (dose group of 20 mg/kg) after the end of study showed increased hematocrit values against starting values. Significant decrease of platelets in males of dose group 20 mg/kg against starting values after 60 and 90 days of the test article application were noticed, at the end of study against control group, too. The decrease of platelets in males of all groups at the end of the study was registered. In satellite males dose group 40 mg/kg the return to the starting values was observed. In females of dose group 20 mg/kg the decrease of platelets after 90 days against starting value was observed. The significant changes in value of MCV, MCH, and MCHC were registered during experiment. The causality with test article was not found. No significant changes in differential count attributable to the administration the test article could be discerned. Significant increase of values Quick test against control group after 90 days in males dose groups 5 and 20 mg/kg were registered. All significant differences in hematology parameters which were found can be considered to be without toxicological importance because so dose related relationship was evident. They can be explaining as the result of intraindividual and interindividual variability, or they have only statistical importance.
CLINICAL CHEMISTRY
In same cases the incidence of blood hemolysis was registered. The results of these samples were not included into the statistical evaluation of clinical chemistry parameters. These results would not positive influence on the correct evaluation. The hemolysed blood samples were registered in control group and dose groups too.
There was some small differences in activity of AST and ALT in animals. These changes have not connection with test article. The significant increased activity of ALP against control group after 60 days of administration of the test article in females doses group 40 mg/kg was registered. Increased activity of ALP in males of all dose groups against control group was registered at the end of the test article administration. Dose dependence is evident. The increase of ALP activity was reversible in males, in females probably longer time is necessary for return on the normal level.
URINALYSIS
The urine analysis was performed at the end the study. No significant changes against normal physiological conditions were detected.In the urine of some animals small amount of protein, ketones and presence of blood was observed ( often considered as physiological). There are no differences between control and dose groups.
In parameter of urine volume( collected during 6 hours) no significant differences between control and dose groups were showed.

NEUROBEHAVIOUR

ORGAN WEIGHTS
A significant decrease of the relative weight of the kidney right was observed in low dose group of males in compare to medium and high dose groups. The relative weights of the both adrenals were significantly decreased in males in the high dose level in compare to low dose group. A significant decrease of the relative weight of kidney right was found in females of medium dose level in compare to control group and a significant decrease of the ovary left was shown in the same group in compare with high dose group. Relative weight of kidney right was increased and weight of the thymus was decreased in males of satellite high dose group in compare to satellite control group. A significant increase of the liver and a decrease of the brain were observed in females of satellite high dose group in compare to satellite control group. There was not evident dose dependence of significant defference of relative organs weight between control and treated animals. Relative weights of the other organs in the treated animals were not significantly different from those in the control group.

MACROSCOPIC EVALUATION
Four rats died during the study: 1/24 of the control group(female), 2/24 of the high dose level (female) and 1/24 of the high dose level (male). Acute diffuse emphysema of lungs was observed in all dead rats during a necropsy. A couse of the death was asphyxia. The test substance did not induce the exitus of animals. Other animals survived until the necropsy. The uterus was enlarged and uterine horns contained a dark fluid in 1/24 female in the control group. A small cyst in the ovary right was observed in 1/24 female No 40 of high dose group.
GROSS PATHOLOGY

HISTOPATHOLOGY: NON-NEOPLASTIC

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)
HISTOPATHOLOGICAL EVALUATIOM
Kidneys: In the kidneys of males and females there were foci of mild hyperemia. This lesion is connected with killing of animals and not adequate bleeding. Interstitial nephritis as a primary inflammatory disease observed in our case, appears to be immunologically induced phenomenon. Immunologically induced interstitial nephritis occurs spontaneously in mice and can be induced in some breeds of rats.
Limph nodes: Lymph nodes of males and females demonstrated hyperplasia of cortex and poracortex. This reaction can be given into connection with observing of nematoda in large intestine.
Liver: incidental findings of lymphoepitelioid granulomas ( males C 1/15, H 1/14) in the liver can be connected with damage of the lower digestive tract and entering of the digestive bacteria the portal circulation. The result can be focal hepatocellular necrosis followed by healing process.
Intestines: Incidentally, parasitic infestation was found in the large intestine (females, males). The eosinophilic of intestinal wall was observed in the small intestine (males). It is known that this infection induce eosinophilic infiltration (females, males) of the mucosal intestine and reaction of the regional lymph nodes. Observed nematoda are included into genus Oxyuridae, which can be found during necropsy without clinical changes.
Lungs: Focal hyperemia of the lungs in males and females is due to mode of killing of animals. Eosinophils (female, males) are commonly present in varying numbers during both the acute chronic inflammatory phases in the lungs under a variety of pathological situations, and the predominance of the cell type is indicative of a hypersensitivity reaction.
Heart: A small focus of myocytes loss in rat with mononuclear cell infiltration represents the healing process (males) following a localized area of necrosis. This appearance is typical of the minor lesions that are commonly encountered as a spontaneous change in animals form toxicology studies.
Spleen: spleen in one case of male demonstrated focal hyperemia due to mode of animals killing(males). Higher occurrence of siderophages was observed in females and in males.
Uterus: As it was seen in both groups, uterus wall of female was infiltrated with eosinophils. It is connected with estrogens regulation.
Thymus: Thymus cysts (female) can be found within the developing and mature thymus. They are usually of no significance.
Skin: Granulomatous response around the hyperplastic sebaceous gland (females) is characterized by number of macropahges and multinucleated giant cells together with active fibroblasts. It sis supposed to be primary infection of the dermis and followed the hyperplasia of the sebaceous glands.
OTHER FINDINGS

Effect levels

Dose descriptor:

NOAEL

Sex:

male/female

Basis for effect level:

other: Doses 5, 20 and 40 mg/kg/ bw/day were used. No adverse effects were observed.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified