2H-Indol-2-one, 1,3-dihydro-1-phenyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 January 2004 to 11 November 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: There are no deviations from the recommended guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

441-020-3

EC Name:

-

Cas Number:

3335-98-6

Molecular formula:

C14H11NO

IUPAC Name:

N-phenylindolin-2-one

Details on test material:

- Name of test material (as cited in study report): Phenyloxindole
- Physical state: yelllowish powder
- Analytical purity: 99.80%
- Lot/batch No.: CHAD 9902K
- Storage condition of test material: at dark and cool

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, TOP VELAZ, 165 00 Praha, Czech Republic
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: males 165 - 231 g, females 126 - 179 g
- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20cm) containing sterilised clean shavings of soft wood.
- Diet (e.g. ad libitum): Complete pelleted diet for rats and mice in SPF breeding (ST 1 BERGMAN), producer: Mill Kocanda, Jesenice by Prague was used. Diet was sterilised before using.
- Water (e.g. ad libitum): Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Water was sterilised before using.
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle.


IN-LIFE DATES: From: 30 January 2004 To: 11 June 2004

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The concentrations of suspensions in all three-dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form (suspension in olive oil) was prepared daily just before administration. The vehicle control group was administered by olive oil in the same volume per 100 g of body weight. The application form of the test substance was prepared daily before administration and during the administration it was mixed by magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability was monitored in olive oil suspension under continual mixing. The mixture was mixed all the time of monitoring. The measurement was performed on two concentration levels (3 and 30 g/L) in 5 time intervals (0, 30; 60 and 120 minutes and 24 hours).

The homogeneity of application form was monitored by the analyses of olive oil suspension in three sampling spots (the bottom, the middle and the surface of container content). Measusement was performed on iwo concentration levels (3 g/L and 30 g/L). The mixture was mixed all the time of monitoring.

Duration of treatment / exposure:

91 days

Frequency of treatment:

Seven days a week

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 100 and 300 mg/kg body weight/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses for the were chosen with respect to the results of 28-day toxicity study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Health condition and general clinical observations, daily.
Behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypies or bizarre behaviour.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly


WATER CONSUMPTION: Yes
- Time schedule for examinations: twice a week


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in acclimatisation period and all animals in the last week of study


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 92nd day
- Anaesthetic used for blood collection: diethyl ether narcosis
- How many animals: all animals
- Parameters:
Total erythrocytes count
Mean corpuscular volume
Haematocrit HGT
Haemoglobin concentration
Total leucocytes count
Total platelet count PLT
Partial thromboplastin time
Prothrombin time
Prothrombin time - INR
Granulocytes
Lymphocytes
Monocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 92nd day
- Anaesthetic used for blood collection: ether narcosis
- How many animals: All animals
- Animals fasted: Yes. The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.
- Parameters:
Glucose
Cholesterol, total
Urea
Bilirubin, total
Aspartate aminotransferase
Alanine amonitransferase
Alkaline phosphatase
Calcium
Phosphorus
Protein, total
Protein, albumin
Creatinine
Sodium
Potassium
Chloride


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the last week of administration
- Dose groups that were examined: alls animals
- Battery of functions tested: During functlonal examination, the sensory reactivity on auditory, papillary reflex, visual and proprioceptive stimuli were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made in 1) pectoral legs, 2) pelvis legs, 3) all four legs.


Biometry of organs:
The absolute weights of liver, kidneys, adrenals, testes or ovaries, epidydimide (only one; the second one was used for sperm observations), thymus, spleen, brain and heart were recorded.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities was carried out. Organs for consequent histopathological exaxnination were taken out and stored in containers with fixative (neutral 9% formaldehyde).

HISTOPATHOLOGY: Yes
Tissue specimens were fixed in 4% neutral formaldehyde (testes and epididymides were fixed in Bouin's solution) processed by routine paraffin technique and stained by hematoxyline-erytrosine. Cryotome sections of liver and kidneys were stained by oil red for neutral lipids. Tissues and organs were collected at the scheduled sacrifice from all animals of the control and the high dose group. Also lungs, liver, kidney, forestomach, stomach, gross lesions, coagulation gland, pituitary gland, prostate gland, seminal vesicles, teste, ovaries, pituitary gland, uterus, cervix uteri, vagina were investigated in medium and low dose group.

Statistics:

The following data were statistically evaluated: haematology, blood chemistry and body weight. The Mann-Whitney U-test at significance level 0.05 was used for statistical analysis. The control group was compared with three treated groups.

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:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study.
In control and exposed animals no clinical changes and no toxic changes were observed during all the study.
MALES and FEMALES: The activity of all animals in all treated groups was during the study similar and unchanging compared with activity of animals in control group. No changes were found out at all dose levels during other examinations: position of eyelids, tonic movements, clonic movements, reaction to handling, lacrimation, salivation, piloerection, emiction, defecation, colour of mucous membranes, vocalisatlon and other activity.

BODY WEIGHT
MALES: The growth curves of all treated groups were analogous with the growth of control group. Only the growth curve of dose level 300 mg/kg proceeds slightly above others.
FEMALES: The growth curves of females of all treated groups were analogous with the growth of females from the control group.

WEIGHT GAIN
MALES: Average body weight increments were similar in all dose levels. Only in dose level 300 mg/kg the body weight increments were slightly higher
FEMALES: Variations of body weight increments are only individual. Average body weight increments of females of all treated groups were generally similar with females of control group.

FOOD CONSUMPTION
MALES: Food consumption of all treated groups of males was similar to control group.
FEMALES: Food consumption of all treated groups of females was lower than in the control group.

WATER CONSUMPTION
MALES: Water consumption was slightly higher in males of the dose level 300 mg/kg. In the dose level 30 and 100 mg/kg the water consumption was analogous to control.
FEMALES: Water consumption of females of the dose level 30 mg/kg was similar with the control group. Water consumption was lower in females of the dose level 100 mg/kg and 300 mg/kg.

OPHTHALMOSCOPIC EXAMINATION
MALES and FEMALES: No changes were found out in all animals.

HAEMATOLOGY
MALES: lncreased values of total leucocyte count and total platelets count were found out in males of all treated groups after application of test substance. Thromboplastin time, prothomboplastin time and PT-INR were increased only at dose levels 100 and 300 mg/kg. These values were evaluated as statistically significant at dose level 300 mg/kg. Increased count of lymphocytes in differential leucocytes count was found out in dose level 300 mg/kg. lncreased counts of total erythrocyte, haemoglobin concentration and haematocrit were found in dose level 100 mg/kg. The values of total erythrocyte count and haemoglobin concentration were statistically significant. Decreased values of haemoglobin concentration, count of granulocytes and monocytes at dose level 300 mg/kg were found out.
FEMALES: Increased values of total leukocyte count, total platelets count, thromboplastin time, prothoniboplastin time and PT-INR count were found out in females after application of test substance. Increased count of lymphocytes in differential leucocytes count was found out only in dose level 100 mg/kg. Increased count of total erythrocyte, haemoglobin concentration and haematocrit was found in dose level 100 mg/kg. Haematocrit value was evaluated as statistically significant. Decreased values of count of granulocytes and monocytes in dose level 100 mg/kg were found out. Changes detected in females of group 30 (increased value of total erythrocytes, haemoglobin concentration and haematocrit) were already shown in males of group 100 mg/kg.

CLINICAL CHEMISTRY
MALES: Increased values were found in the case of glucose, total cholesterol, urea in dose level 30 and 100 mg/kg, total bilirubin, total protein, phosphorus and potassium in dose level 300 mg/kg. Decreased values of AST, ALT and ALP were found in dose level 300 mg/kg. Even if these differences are statistically significant (e.g. albumin, sodlum and chloride), the changes were of no biological significance.
FEMALES: Increased values of glucose, cholesterol, ALP and total protein in all dose levels were found, increased value of urea was found out only in dose levels 100 and 300 mg/kg. Decreased values of AST and creatinine were found out in all dose levels. Although some significant differences were shown in statistical evaluatlon (e.g. phosphorus), for biological aspect these parameters had shown no important differences.

NEUROBEHAVIOUR
MALES: Reaction to noise, reaction to pain and papillary reflex were same in treated as in the control group. Upstanding, emiction and defecation results were not equal to the control, but they were in the range of physiological reaction of animals. All values of grip strength (pectoral, pelvis and total) were analogous in the treated and control groups.
FEMALES: Reaction to noise, reaction to pain and papillary reflex were same as in the control group. Upstanding, emiction and defecation results were not equal to the control, but they were in the range of pliysiological reaction of animals. The values of upstanding of females of all groups (including females of control group) were markedly higher in relation to males. All values of grip strength (pectoral, pelvis and total) were analogous in the treated and control groups.

ORGAN WEIGHTS
MALES: The weight (absolute and relative) was increased in kidneys, liver and prostate gland at dose level 300 mg/kg.
FEMALES: Increased weight (absolute and relative) was found in liver and uterus at dose levels 100 and 300 mg/kg; spleen at dose level 300 mg/kg only.

GROSS PATHOLOGY
MALES: During the macroscopic examination of males no important pathological changes were found out. Examinatlon of the externa1 surface of the body revealed no changes. Incidental findings included discolouration of kidneys and liver, oedema of mucosa of stomach, dark red foci on the thymus were recorded. In the absence of a treatment-related distribution they were considered as changes of no toxicological significance.
FEMALES: During the macroscoplc examination of cranial and thoracic tissues and organs of females no important pathological changes were found out. Only uterine horns filled by pellucid liquid were found out in abdominal cavity. Examination of the external surface of the body revealed no changes. Fluid in the uterus found in females of dose level groups 30, 100 and 300 mg/kg/day is related to the stage of oestrous cycle and it is a normal finding.

HISTOPATHOLOGY:
Incidence of affected animals is expressed in numeric form and ranged in sequence of dose levels 0-30-100-300 further in the text.
MALES: Various alterations were registered in liver in all treated groups. Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in Iiver parenchyma) was observed in 3-5-4-3 males. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 6-5-1 -3 males. Centrilobular steatosis (increased amount of lipids in hepatocytes of centrilobular area) was detected in 1-0-5-4 males. Special histological staining for neutral lipids manifested fatty changes.
Syphacia muris in the lumen was found in 2-4-1-0 males at microscopical examination of the colon. Metastatic mlneralization in kidney (basophilic concrements in tubules of corticomedullary area without reaction of surrounding tissue) was recorded in 0-0-2-0 males. Steatosis of kidneys cortex was found in 5-5-6-6 males. Atrophy of germinal epithelium of testes was found out in 2-1-0-0 males and oedema of testes in 3-0-0-0 males was recorded. Histopathologlcal findings in prostate gland were more often: focal inflamation of interstitium in 3-2-2-3 males and focal hyperplasia in 1- 1-0-0 male.
FEMALES: Various alterations were registered in liver in all treated groups. Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in liver parenchyma) was observed in 7-4-7-6 females. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 3-6-2-0 females. Centrilobular steatosis (increased amount of lipids in hepatocytes of centrilobular area) was detected in 0-0-
1-4 females. Special histological staining for neutral lipids manifested fatty changes. Syphacia muris in lumen was found in 1-2- 1 - 1 females at microscopical examination of the colon. Further histopathological affections were found: Changes in kidneys - metastatic calcification = mineralization (basophilic concrements in tubules of corticomedullary area without reaction of surrounding tissue) was the most often finding in 7-9-8-9 females. Steatosis of kidneys cortex was found in 6-5-9-9 females, pyelitis and urolithiasis in 0-0-1-0 female. In female genital tract the following sporadic affections were detected: ovarian follicular cyst 0-0-1-0, uterus - metaplasia of endometrium 1-0-0-0, uterus inflammation 1-0-0-0 and uterus dilatation 0-1 -0-1.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

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:

The series of effects observed in haematological, biochemical and pathological examination at the dose levels 30 and 100 mg/kg/day could not be denoted as negative effect of biological significance. As a negative effect of the test substance it is possible to consider centrilobular steatosis of liver at dose level 300 mg/kg/day.
Based on the results, the NOAEL for the 90-day study was established as 100 mg/kg body weight/day.

Executive summary:

The test substance, Phenyloxindole, was tested in combined Repeated Dose 90-day Oral Toxicity/One-Generation Reproduction Study in rats. For the 90-day toxicity part of study 60 male rats and 40 female rats were used (15 males and 10 females for each dose level). Wistar rats of SPF quality were used for testing. The test substance was administered in olive oil by stomach tube; oral appllcation was made daily. The 4 groups of animals were included in the study - 3 treated groups (doses 30, 100 and 300 mg/kg body weight/day) and one control group (vehicle only). The clinical observation and health status control were performed daily. The body weight, food consumption were measured weekly as well as the detailed clinical observation was carried out. Water consumption was measured twice a week. Before the end of study the functional observation battery was accomplished. The study was finished by haematological and biochemical analysis, ophthalmology examination and gross necropsy of animals. The selected organs for weighing and histopathology examination were removed. The series of effects observed in haematological, biochemical and pathological examination at the dose levels 30 and 100 mg/kg/day could not be denoted as negative effect of biological significance. At dose level 300 mg/kg/day increased APTT, PT, potassium, weight of liver, kidney and prostate gland, and decreased values of AST, ALT, ALP in males were recorded. In females decreased total erythrocyte count, haematocrit and value of AST and increased values of APTT, PT and PT-INR, protein total, weight of liver, spleen and uterus and were recorded. Centrilobular steatosis in liver of both sexes was observed at histopathological examination. Based on the results of the study, the NOAEL was established as 100 mg/kg body weight/day.