Sodium 4-oxovalerate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 03rd to December 28th, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

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)

Version / remarks:

Adopted 25th June 2018

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)

Version / remarks:

Published in O.J. L142, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar CRL (SPF quality – guaranteed, delivered by Charles River via VELAZ )

Details on species / strain selection:

laboratory rat has been chosen because the testing laboratory has long experience with this species

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Lysolajské údolí 15/53, 165 00, Prague 6, Czech Republic, RČH CZ 11760500
- Age at study initiation: 7 weeks
- Weight at study initiation: 247,75 (mean weight)
- Housing: The study was performed in an IVC TOUCH SLIME LINE animal house of CETA in SPF (Specified Pathogen Free) conditions according to internal SOP No. 217. Animals were housed in the animal room, 2 rats of the same sex in one plastic cage (polycarbonate cages for rats TECNIPLAST, type GR 900 Italy)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Complete pelleted diet for rats – Altromin 1321; Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany.
Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Ministry of Health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 – 70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle (according to internal SOP No. 46)

IN-LIFE DATES: From: 31.03 - 03.04.2021 To: 28.06 - 01.07.2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

aqua pro iniectione

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulation was prepared by mixing with Aqua pro iniectione. Two concentrations of test item formulation were prepared (1 000 mg/10 mL and 10 mg/10 mL).

Concentration level 10 mg/10 mL:
Ca 100 mg (ca 120 µL) of delivered chemical was weighed using Pasteur pipette into narrow 150 mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle. The solution was stirred by magnetic stirrer at 300 rpm (3.5 cm stirrer) for 30 minutes.

Concentration level 1000 mg/10 mL:
Ca 10 g (ca 12 mL) of delivered chemical was weighed using Pasteur pipette into narrow 150 mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle. The solution was stirred by magnetic stirrer at 300 rpm (3.5 cm stirrer) for 45 minutes.

VEHICLE
Supplier: Manufacturer Ardeapharma Ševětín, Czech Republic
DRFE:
Batch No.: 2005110305, Exp.: 05/2022
Batch No.: 2004280281, Exp.: 04/2022
Main study:
Batch No.: 2101070010, Exp.: 01/2024
Batch No.: 2101120024, Exp.: 01/2024
Batch No.: 2102090110, Exp.: 02/2024
Batch No.: 2103190220, Exp.: 03/2024

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item stability and homogeneity were determined by means of measuring of peak area of test item by high-performance liquid chromatography (HPLC) based on a method developed for the purpose of this study at the test facility.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 days per week at the same time (8.00 – 10.00 am)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

Basic groups: 10 per sex per group

Satellite groups: 6 per sex per group (control and 1000 mg/kg bw)

Control animals:

yes

Details on study design:

- Dose selection rationale: A dose-range finding experiment (DRFE) was performed to determine the dose levels for the main study. The following examinations was performed in DRFE: body weight, clinical examinations, haematological examination, pathological examination and biometry of some organs of animals. Results were served for establishing the doses for main study.
- Rationale for selecting satellite groups: The administration period lasted 90 days. Animals in the main groups were then sacrificed, while satellite animals were observed for the next 28 days without treatment.
- Post-exposure recovery period in satellite groups: 28 days.
- Dose range finding studies: The 28-days dose-range finding experiment was performed. In the dose-range finding experiment with test item acid no animal died during the application period due to the toxicity of the test item. Clinical observation did not detect the impact of the test item on the health condition of animals at all dose levels. Slight changes of body weight and body weights increments were detected but without toxicological importance. The administration of the test item did not induce treatment-related toxicologically significant changes.No macroscopical changes related to the test item toxicity were observed during necropsy of treated animals. Biometry of organs did not show treatment related changes in absolute and/or relative weight of organs of treated groups of animals in comparison with the control group of animals. On the basis of the results given above the following dose levels – 0, 100, 300 and 1000 mg/kg bw/day were proposed for the main Repeated Dose 90-day Oral Toxicity Study.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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 (if drinking water study): Yes
- Time schedule for examinations: three times a week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the 1st week of study and at the last week of administration
- Dose groups that were examined: control group, high dose group and in both satellite groups (control and 1000 mg/kg bw/day)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 91st (main groups); 119th (satellite groups) day of study
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, for 18 hours before blood collection but they were supplied with drinking water ad libitum
- How many animals: Not specified
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 91st (main groups); 119th (satellite groups) day of study
- Animals fasted: Yes, for 18 hours before blood collection but they were supplied with drinking water ad libitum
- How many animals: Not specified
- Parameters checked in table [No. 2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 90th (main groups); 118th (satellite groups) day of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No. 3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the last week of administration and recovery period
- Dose groups that were examined: all
- Battery of functions tested: sensory reactivity on auditory, visual, proprioceptive stimuli / pupillary reflex / grip strength / motor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No. 1)

HISTOPATHOLOGY: Yes (see table No. 1)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Health Condition Control
Males
In control males and treated males of all dose levels no signs of disease were recorded during the check-in, acclimatisation and application period. During the observation period no changes of health status were noted in treated and control males. Hindered breathing and piloerection were observed in one male at the dose level 1 000 mg/kg bw/day from the 89th to 90th day (at the 13th week) of the aplication period.
Satellite males
In control males and treated satellite males no signs of disease were recorded during the check-in, acclimatisation and application period. During the observation period no changes of health status were noted in the control and the satellite treated males.
Females
In control females and treated females of all dose levels no signs of disease were recorded during the check-in, acclimatisation and application period. During the observation period no changes of health status were noted in the treated and the control females.
Satellite females
In control females and treated satellite females no signs of diseases were recorded during the check-in, acclimatisation and application period. During the observation period no changes of health status were noted in the treated satellite females and the control satellite females.

Clinical Observations
Males
No clinical changes were recorded in control and treated males during the application period, except one male at the dose level 1 000 mg/kg bw/day. In this male hindered breathing and piloerection were observed at the 13th week of the application period.
Satellite males
No clinical changes were recorded in the control and the satellite treated males.
Females
In control females and treated females no signs of diseases were recorded during the application period.
Satellite females
No clinical changes were recorded in control and treated satellite females during the application period.

Detailed Clinical Observation
Males
The behaviour and activity of all males of all treated groups were similar during the study and not different from males of the control group. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of treated males. Hindered breathing and piloerection were observed in male No. 42 at the dose level 1 000 mg/kg bw/day from the 89th day to 90th day (13th week) of the application period.
Satellite males
The behaviour and activity of all satellite males in the treated group was similar during the whole study and not different from satellite control males.
Females
The behaviour and activity of all females of all treated groups were similar during the study and not different from females of the control group. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of treated females.
Satellite females
The behaviour and activity (poise, gait, reaction to handling) of all females in the treated group was similar during the study and not different from satellite control females.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during all the study.

Description (incidence and severity):

100 mg/kg bw/day
The body weights of males were slightly decreased compared to the control group for the whole administration period. Body weight increments were similar or slightly decreased in males compared to the control group for the whole application period. The body weight increments were variable in females during the whole application period. The total body weight increments of females were increased compared to the control group.

500 mg/kg bw/day
The body weights of males were slightly decreased and body weight increments were similar or slightly decreased in males for the whole application period. The total body weight increments of females were increased compared to the control group.

1 000 mg/kg bw/day
The body weights of males were slightly decreased and body weight increments were similar or slightly decreased for the whole application period. Minimal negative body weight increment was recorded in males at the 13th week of application The total body weight increments of females were increased.

Satellite animals
Decreased body weights and necropsy body weights were detected in satellite treated males for the whole application and recovery periods. The body weights and necropsy body weights of satellite treated females were similar or slightly higher in comparison with the control satellite females during the whole application period and recovery period. Negative body weight increments were recorded in satellite treated females at the 8th week of study. Body weight increments were slightly decreased in satellite males for the whole application and minimal negative body weight increment was recorded at the 15th week – recovery period.

Description (incidence and severity):

Food Consumption in gavage study
Males
Food consumption of treated males was similar compared to the control males during the whole application period.
Satellite males
Food consumption of treated satellite males was comparable with the control males for the whole application and recovery periods.
Females
Food consumption of treated females was comparable with the control females during the whole application period.
Satellite females
Food consumption of treated satellite females was comparable with the control satellite females during the whole application period and recovery periods.

Food Conversion
Males
The food conversion of treated males was variable during application period compared to the control group.
Satellite males
The food conversion of treated males was variable during application and recovery periods compared to the control group.
Females
An imbalance in food conversion was recorded at all treated groups and the control group during the whole application period. This difference was not dependent on dose level.
Satellite females
An imbalance in food conversion was recorded at treated group and the control group during the whole application period and recovery periods

Description (incidence and severity):

Water consumption in gavage study
Males
Water consumption of treated males was similar compared to water consumption of control males during the whole application period.
Satellite males
Water consumption of satellite treated males was slightly decreased compared to the satellite control males during the application and recovery periods.
Females
Water consumption of treated females was comparable with water consumption of control females during the whole application period.
Satellite females
Water consumption of satellite treated females was similar comparison with satellite control females during the application and recovery periods.

Description (incidence and severity):

The ophthalmologic examination was performed in all animals at the beginning of the study. An ophthalmologic examination was also performed at the end of the study in animals of the control group, high dose group and in both satellite groups (control and 1000 mg/kg bw/day). No changes related to application of the test item were observed in the 1000 mg/kg bw/day group (highest dose level); therefore, animals of lower dose groups were not examined.
Males
During an examination performed before the start of the study and at the end of the study no changes were noted.
Satellite males
During an examination performed before the start of the study no changes were noted.
Females
During an examination performed before the start of the study and at the end of the study no changes were noted.
Satellite females
During an examination performed before the start of the study no changes were noted

Description (incidence and severity):

Males
No significant differences were recorded in paramaters of white blood components.
Examination of haemacoagulation parameters showed statistically significant decreased value of APTT - in males at the dose level 100 mg/kg bw/day. Statistically significant decrease of representation of PLT was recorded in treated males at the dose levels 500 and 1 000 mg/kg bw/day. Statistically significant increase of percentage representation of RT were observed in all treated males.
All haematological parameters were in a range of historical control.

Satellite males
Statistically significant decrease of percentage representation of NEU and statistically significant increase of percentage representation of LYM were noted in satellite treated males. Other haematological parameters of satellite treated males were similar comparison with the satellite control males.
All haematological parameters were in a range of historical control

Females
The statistically significant difference was recorded in white blood components - the value of WBC was increased in females at the dose level 1 000 mg/kg bw/day and percentage representation of LYM was increased in females at the dose level 100 mg/kg bw/day.
Statistically significantly decreased values of APTT and PT in females at the dose level 500 mg/kg bw/day were detected. Statistically significant decrease of value of FIB in females at the dose level 100 mg/kg bw/day was recorded during examination of haemocoagulatuion parameters.
Statistically significant increase of percentage representation of RT were noted in treated females at the dose levels 100 and 500 mg/kg bw/day.
All haematological parameters were in a range of historical control.

Satellite females
All haematological parameters of satellite treated females were similar comparison with the satellite control females.
All haematological parameters were in a range of historical control.

Description (incidence and severity):

Males
Statistically significant differences were recorded in treated groups of males.
At dosed group 1 000 kg/kg bw/day statistically significantly decreased values of T-Pro, BUN, ALB and concentration of Ca ions and increased values of TG and T-BIL were recorded.
Statistically significantly increased value of TG was recorded at dosed group 500 mg/kg bw/day compared to control group. Statistically significantly decreased value of BUN was recorded at dosed group 100 mg/kg bw/day compared to control group. Values of other biochemical parameters of treated males were quite balanced to the control group. All biochemical parameters were in a range of historical control.

Satellite males
Statistically significantly increased concentration of inorganic IP was recorded in treated males. Other biochemical parameters of satellite treated males were similar with the satellite control males.
All biochemical parameters were in a range of historical control.

Females
Statistically significant differences were recorded in treated groups of females.
At dosed group 1 000 mg/kg bw/day statistically significantly decreased values of T-Pro, BUN, ALB, CHE and concentration of Cl ions were recorded. Statistically significantly decreased value of CHE and of Cl ions were recorded in females at the dose level 500 mg/kg bw/day compared to control group. Decreased value of CHE at the dose 1 000 mg/kg bw/day was irreversible.
Statistically significantly increased value of Na ions was recorded in females at the dose level 100 mg/kg bw/day compared to control group.
Values of other biochemical parameters of treated males were quite balanced to the control group. All biochemical parameters were in a range of historical control.

Satellite females
Statistically significantly decreased values of ALP, CHE and K ions were recorded at satellite treated females in comparison with the control females.
Values of other parameters of satellite treated females were similar to satellite control females.
All biochemical parameters were in a range of historical control.

Description (incidence and severity):

Males
Slightly increased concentration of TSH hormone in treated males at the dose level 1 000 mg/kg bw/day was detected.
Statistically significant differences in concentrations of T3, T4 and TSH hormone were not detected.
Mean concentrations of T3, T4 and TSH hormone were similar in treated males in comparison with the control males.
All parameters were in a range of historical control.

Satellite males
Statistically significant differences in concentrations of T4, T3 and TSH hormone were not detected.
Mean of concentrations of T4, T3 and TSH hormone were similar in treated males in comparison with the control males.
All parameters were in a range of historical control.

Females
Statistically significant differences in concentrations of T4, T3 and TSH hormone were not detected.
Mean of concentrations of T4, T3 and TSH hormone were similar in all treated females in comparison with the control females.
All parameters were in a range of historical control.

Satellite females
Statistically significant differences in concentrations of T3, T4 and TSH hormone were not detected.
Mean of concentrations of T3, T4 and TSH hormone were similar in satellite treated females in comparison with the satellite control females.
All parameters were in a range of historical control.

Description (incidence and severity):

Males
Statistically significant differences in volume of urine were not detected. Statistically significant decreased pH of urine was detected in treated males of doses 500 mg/kg bw/day and 1 000 mg/kg bw/day.
Presence of proteins (2 – 1 – 5 – 1 males) and blood (1 – 0 – 0 – 0 males) were recorded in treated males as well as in control males.
Presence of ketones (0 – 0 – 7 – 5 males) and leukocytes (0 – 1 – 1 - 1 males) were recorded in treated males.
The values of all other parameters of treated males were similar to control.

Satellite males
Statistically significant differences in pH and volume of urine were not detected. Presence of protein in urine was recorded in 2 - 2 males. Presence of blood in urine and of urobilinogen was recorded only in 0 - 1 satellite male. Presence of leukocytes in urine was observed in 1 - 4 males. The values of all other parameters of treated males were similar to control.

Females
Statistically significantly decreased pH of urine was detected in treated females of dose level 500 mg/kg bw/day. Statistically significantly decreased volume of urine was detected in treated females of all doses.
Presence of proteins (0 – 0 – 0 - 1 female), ketones (0 – 0 – 1 – 7 females), leukocytes (1 – 0 – 2 - 1 females) blood (0 – 1 - 0 - 0 female) and of urobilinogen (0 – 0 – 0 – 1 female) were recorded in control and treated females.

Satellite females
Statistically significant differences in volume of urine and pH of urine were not detected. Occurrence of leucocytes in urine in 1 - 3 satellite females was recorded. The values of other parameters of treated satellite females were similar to the control satellite females.

Description (incidence and severity):

Males
Reactions to touch, noise, pain and pupillary reflex of treated males were the same as in the control group. The number of emiction and defecation of treated animals were without important changes. The number of upstanding in males at the dose level 100 mg/kg bw/day was increased compared to the control group. The variability was adequate to species, sex and age of animals used in experiments.
In male No. 42 at the dose level 1 000 mg/kg bw/day grip strength were not examined for unhealthy condition. Reactions to touch, noise and pain of the male No. 42 was decreased.
Satellite males
The activity (poise, gait, reaction to handling) of all males in the satellite treated group was similar during the study and there was no difference in the activity of males in the satellite control group.
Females
Reactions to touch, noise, pain and pupillary reflex of treated females were the same as in the control group. Emiction and defecation of treated animals was without importatnt changes. The variability was adequate to species, sex and age of animals used in experiments.
Satellite females
The activity (poise, gait, reaction to handling) of all females in the satellite treated group was similar during the study and there was no difference in the activity of females in the satellite control group.

Immunological findings:

not examined

Description (incidence and severity):

Absolute Organ Weight

Males
Absolute weight of the seminal vesicles with coagulating glands at the dose level 1 000 mg/kg bw/day was statistically significantly decreased compared to the control group.
Absolute weights of other organs of treated males were balanced with control males.

Satellite males
Absolute weights of all organs of satellite males were balanced with control males. Statistically significant changes in absolute organ weights of satellite males were not detected.

Females
Absolute weight of the brain at the dose level 100 mg/kg bw/day was statistically significantly decreased compared to the control group. Absolute weights of other organs of treated females were balanced with control males.

Satellite females
Absolute weights of the heart and pituitary gland were statistically significantly increased compared to the control group. Absolute weights of other organs of satellite females were balanced with control males.


Relative Organ Weight

Males
Statistically significantly increased relative weights of the kidneys in males at the dose levels 500 mg/kg bw/day and 1 000 mg/kg bw/day were recorded. The relative weight of other organs of treated and control males were similar.

Satellite males
Statistically significantly increased relative weight of the adrenal glands in satellite treated males was recorded. The relative weight of other organs of satellite treated and control males were similar.

Females
Statistically significantly decreased relative weight of the brain at the dose levels 100 mg/kg bw/day and 500 mg/kg bw/day in treated females were recorded. The relative weight of other organs of treated and control males were similar.

Satellite females
Statistically significantly increased relative weight of the kidneys and heart in satellite treated females were recorded. The relative weight of other organs of satellite treated and control females were similar.

Description (incidence and severity):

Macroscopic Findings

Males
No macroscopical findings were recorded in 9 – 9 – 10 – 9 males during necropsy. Reduced testes with ragged consistency and reduced epididymis were found in one treated male – 100 mg/kg bw/day and in one control male. Dilated esophagus, flatulence in stomach and in intestines, no intestinal contens in large and small intestines, black content in caecum and hemorrhages of mesenteric lymph nodes were found in one treated male (No. 42) at the dose level 1 000 mg/kg bw/day.

Satellite males
No macroscopical findings were recorded in 6 - 6 males during necropsy.

Females
No macroscopical findings were recorded in 9 – 10 – 10 – 10 females during necropsy. Cysts of right ovary was found in one control female. Non-pathological finding – uterus dilatation in 2 – 3 – 2 – 4 females was detected.

Satellite females
No macroscopical findings were recorded in 6 – 6 satellite females during necropsy.

Neuropathological findings:

not examined

Description (incidence and severity):

The full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs with macroscopical changes in five females (uterus: dilatation) and in one male (testes and epididymis: reduced) were examined at the lowest and middle dose level groups.
Males
The histopathological examination organs in males showed only sporadical microscopical changes and these changes did not related with the test item treatment.

Satellite males
The histopathological examination organs in satellite males showed only sporadical microscopical changes and these changes did not related with the test item treatment.

Females
The histopathological examination organs in females showed only sporadical microscopical changes and these changes did not related with the test item treatment.
The hydrometera is a non-inflammatory dilatation of the uterus with secretion, normally detected at the end of the proestrus and the beginning of the oestrus as side affect of the ovarian cycle.

Satellite females
The histopathological examination organs in satellite females showed only sporadical microscopical changes and these changes did not related with the test item treatment.

Histopathological findings: neoplastic:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects observed at the highest dose tested

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

No adverse treatment-related response that results in change in morphology, physiology or growth of an animal organism was recorded.

The NOAEL (No Observed Adverse Effect Level) value for REPEATED DOSE TOXICITY (90 days) in male and female rats for the test item was established as 1 000 mg/kg body weight/day.

Executive summary:

The test item was tested for subchronic toxicity using the OECD Test Guideline No. 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, Adopted 25^th June 2018.

Methods

Wistar rats of SPF quality were used for testing. The test item was administered in a vehicle (aqua pro iniectione) by stomach tube; oral application to rats occurred daily.  The study includes four main groups and two satellite groups of animals. Each main group consisted of 10 males and 10 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 100, 500, 1 000 mg/kg of body weight/day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1 000 mg/kg bw/day). The administration period lasted 90 days. Animals in the main groups were then sacrificed, while satellite animals were observed for the next 28 days without treatment.   

During the 90-day study clinical observation and health status control were performed daily. The body weight, food consumption were measured weekly and the detailed clinical observation was carried out in the same time interval. Water consumption was measured three times a week. Ophthalmologic examination was performed at the beginning and at the end of the study. Before the end of study the functional observations were performed. The study was completed by urinalysis, haematological and biochemical analysis, and gross necropsy of animals. The organs selected for weighing and histopathological examination were removed. These parameters (except clinical observation during the recovery period) were also checked for the satellite groups of animals.

Results

The oral administration of the test substance to rats by gavage for a period of 90 consecutive days did not cause mortality related with the test item treatment.

No effect of the test item on the body weight, food consumption and water consumption were recorded during the study. Slight changes of these parameters were without toxicological importance.

No clinical findings revealed influence of the test item on clinical status of treated animals and satellite treated animals were recorded.

No toxicologically significant findings were detected during the histopathological examination of organs in animals at the highest dose level and satellite treated animals. The changes found in the organs of control and test animals are, in most cases, spontaneous or agonal changes, which are commonly detected at different frequencies in rats. The observed changes are few and are not caused by the test item. Sporadic findings recorded in adrenal glands, heart, liver, lungs, testes and thymus were not related with test item treatment. Focal ulceration and focal subacute polymorphonuclear inflammation of the esophageal muscle layer are propably caused by esophageal injury during esophageal tube insertion. Other observed changes occur in control and test animals are inconsistent and few in number with no apparent relationship to the effect of the item.

No signs of systemic toxicity were found.