2-[2-(3-butoxypropyl)-1,1-dioxo-1,2,4-benzothiadiazin-3-yl]-5'-tert-butyl-2-(5,5-dimethyl-2,4-dioxo-1,3-oxazolidin-3-yl)-2'-[(2-ethylhexyl)thio]acetanilide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Feb - 24 Mar 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): UY-330
- Physical state: solid
- Appearance: white powder
- Batch No.: CE-201
- Expiration date of the lot/batch: 01 Jan 2005
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

other: Wistar Crl:(WI) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 173 - 202 g (males); 143 - 168 g (females)
- Housing: Animals were housed in groups of 5 per sex in stainless steel cages suspended with wire mesh floors (55 x 34 x 21.5 cm height). During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type III, height 15 cm) with sterilised sawdust provided as bedding.
- Diet: Altromin standard pelleted laboratory animal diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 23.6
- Humidity (%): 44 - 81
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 25 Feb 2004
To: 24 Mar 2004

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity: 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4-5 h prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was chosen based on trial formulations of the laboratory.
- Amount of vehicle (if gavage): 5 mL/kg bw (calculated weekly according to the latest body weight)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance formulations in propylene glycol were noted as stable for at least 4 h and formed a homogeneous suspension at the concentrations tested. The analysis of the accuracy of dose preparations revealed values within the range of 92 - 102% of nominal, which was considered to represent an acceptable level of accuracy.

Duration of treatment / exposure:

28 d

Frequency of treatment:

once daily, 7 d/week

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150 and 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of a 5 d dose range finding study (study report no. 400365) with 3 animals and doses of 150 and 1000 mg/kg bw/d.

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 1, 8, 15, 22 and 28

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 28, prior to scheduled termination
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, overnight
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 28, prior to scheduled termination
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, overnight
- How many animals: all animals

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes with optic nerve and Harderian gland, Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, Larynx, Lacrimal gland, exorbital, Liver, Lung infused with formalin, Lymph nodes (mandibular, mesenteric), Nasopharynx, Oesophagus, Ovaries, Pancreas, Peyer’s patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, Tongue, Trachea, Urinary bladder, Uterus, Vagina, all gross lesions

HISTOPATHOLOGY: Yes
Adrenal glands, Aorta, Brain, Caecum, (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, Lymph nodes, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer’s patches if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), all gross lesions
All collected tissues and organs of the control and the high-dose group and the gross lesions of all animals were examined. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement.

Other examinations:

ORGAN WEIGHTS
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) was applied for the comparison of the treated groups and the control groups for each sex. The Student’s t-test was applied for motor activity data.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

but not related to the test substance

Mortality:

mortality observed, treatment-related

Description (incidence):

but not related to the test substance

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

but not related to the test substance

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

but not related to the test substance

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

but not related to the test substance

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

but not related to the test substance

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

but not related to the test substance

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality and no test substance related clinical signs occured during the study period. The clinical signs were observed in one to three animals of the low and mid-dose males and the control and high-dose females and included laboured respiration, gasping, rales, alopecia, scabs, salivation and a missing tail apex. These findings are more frequently noted in rats of this age and strain and were not considered to be of toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
No effects on body weight and body weight gain were observed during the treatment period.

FOOD CONSUMPTION
There was no difference between food consumption of treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. The statistically significant changes observed included higher relative Iymphocyte counts in males at 50 mg/kg bw/d and lower white blood cell counts and platelet counts in females at 150 mg/kg bw/d. These changes were considered to have occurred by chance and in the absence of a dose related response to be of no toxicological significance.

CLINICAL CHEMISTRY
The clinical biochemistry parameters were considered to have been unaffected by treatment. The observed alterations of the clinical parameters included an increased glucose level measured for high dose males due to the presence of slighty higher values in two males. Since the mean was well within the range of values for this type of study (no further information given in the study report), this change was not considered to be significant in toxicological terms. Other statistically significant changes included lower calcium levels in males at 150 mg/kg bw/d, lower total protein levels in males at 50 mg/kg bw/d and higher, lower albumin levels in males at 50 and 1000 mg/kg bw/d and lower inorganic phosphate levels in females at 50 mg/kg bw/d. These changes did not show a treatment-related distribution and means were within the normal range expected for this type of study (no further information given in the study report). The deviations were considered to have occurred by chance and therefore, no toxicological significance was attributed to these deviations.

NEUROBEHAVIOUR
No differences were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the treated animals, when compared to control animals. Only in the motor activity a variation was observed that did not indicate a relation with test substance treatment.

ORGAN WEIGHTS
No toxicologically significant changes were noted regarding absolute and relative organ weights. In low dose males, a statistically significant lower relative kidney weight was considered not to be a sign of toxicity since no dose-response relationship was observed and the mean was within the range of the control group.

GROSS PATHOLOGY
The necropsy did not reveal any toxicologically relevant alterations. Incidental findings among control and/or treated animals included diaphragmatic hernia of the liver (1/5 males at 150 mg/kg bw/d and 1000 mg/kg bw/d), reduced size of the testes and epididymides (1/5 males at 1000 mg/kg bw/d, confirmed microscopically by testicular and epidydimal agenesis), red foci on the thymus (1/5 males at control and 1000 mg/kg bw/d), red discolouration of the thymus (1/5 males at 1000 mg/kg bw/d), enlarged mandibular lymph nodes (3/5 males and 1/5 females at control, 1/5 males at 50 mg/kg bw/d, 3/5 males at 150 mg/kg bw/d, 3/5 males at 1000 mg/kg bw/d), a missing tip of the tail (1/5 males at 150 mg/kg bw/d) and fluid in the uterus (4/5 females at control, 2/5 females at 50 mg/kg bw/d, 3/5 females at 1000 mg/kg bw/d). These findings are occasionally seen among rats used in these types of studies (no further information given in the study report). In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL corresponds to the highest dose tested

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No systemic toxicity was observed up to a dose of 1000 mg/kg bw/d in male and female Wistar rats during a 28 d-treatment period.