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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from June 4, 2007 to March 30, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Humic acids, sodium salts
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 9. 5. 2007/R
- Expiration date of the lot/batch: 05/2022
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Kolec u Kladna, Czech Republic
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: cca 183-184 g (males), cca 155 g (females)
- Housing: All the study proceeded in SPF (Specified Pathogen Free) animal house.
2-3 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood.
- Diet: ad libitum. Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN). Diet was sterilised before using.
- Water: ad libitum. Drinking water. Water was sterilised before using.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Relative humidity: of 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered dissolved in water for injection
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The vehicle control group was administered by water for injection in the same volume. The application form (test substance solution in water for injection) was prepared daily before administration. These solutions were mixed for 5 minutes by magnetic stirrer.

VEHICLE
- Concentration in vehicle: The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
- Lot/batch no.: Aqua pro injectione. Manufacturer: Ardeapharma a.s., Ševětín, Batch No.: 0102250407, exp.: 4/09

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and the homogeneity of application form were determined.
The stability of application form was monitored by the analyses of solution of test substance in water. The measurement was performed on two concentration levels (250 and 1000 mg/10mL). The solution was prepared by mixing of test substance and water for 5 minutes (cca 400 rpm) in a container for application form preparation. For the determination of stability the samples were taken from the middle of container content after required time intervals (0, 30, 60 and 120 minutes). Time interval 0 min. was the time after 5 minutes mixing. The solution was mixed all the time of monitoring.

The homogeneity of application form was monitored by the analyses of solution of test substance in water prepared by the same way as for the determination of the stability. The measurement was performed on two concentration levels (250 and 1000 mg/10mL). The samples were taken after 5 minute mixing from 3 given places - the bottom, the middle and the surface of container content.

The determination of test substance was performed on the basis of determination of absorbance of a water solution. Test substance stability and homogeneity were determined by measuring of an absorbance of water solution (application form) in visible range of spectrum.
Duration of treatment / exposure:
28 days

Frequency of treatment:
The animals were treated 7 days per week at the same time.

Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses for the 28-day study were chosen with respect to the results of two studies performed at test facility before:
Study No. 27/07/7-P: Humic acids, sodium salts - Repeated Dose (28 days) Toxicity (Oral) - Dose-range finding study.
Study No. 27/07/1: Humic acids, sodium salts - Acute Oral Toxicity - Acute Toxic Class Method
- Rationale for animal assignment: Animals were randomly divided into the control and test groups and marked individually.
- Post-exposure recovery period in satellite groups: 14 days after the end of application
- Section schedule rationale: all animals were sectioned
Positive control:
no

Examinations

Observations and examinations performed and frequency:
HEALTH OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour.
The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of animals was recorded on automatic balances with group average computing module. All animals were weighed immediately before euthanasia too.
Weight increment was computed as an average per group per day (in grams).

FOOD CONSUMPTION: Yes
In the given day of every week the remainder of pellets of each cage was weighed, the new food was weighed out and the food consumption for the previous week was computed.
The average values in groups were calculated for each week of the study. Food consumption for animal/day was calculated from average values of each group. Calculation of food conversion in %: weight increment/food consumption x 100.

WATER CONSUMPTION: Yes
The drinking water consumption was recorded. The average values in groups (water consumption per animal and per day) were calculated for each week of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 29th (main groups) and 43nd day of study (satellite groups)
- Anaesthetic used for blood collection: light ether narcosis
- How many animals: all animals
- Parameters checked in Table 2 were examined.
The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation systems.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 29th (main groups) and 43nd day of study (satellite groups)
- How many animals: all animals
- Parameters checked in Table 3 were examined.
The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.
The blood samples were collected from the orbital plexus under the light ether narcosis. Samples were centrifuged 10 minutes. Biochemical parameters were measured in serum.

URINALYSIS: Yes
- Time schedule for collection of urine: 28th (main groups) and 42nd day of study (satellite groups)
- Metabolism cages used for collection of urine: Yes
The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100g of body weight by gavage to the stomach.
- Parameters checked in Table 4 were examined.

FUNCTIONAL OBSERVATION: Yes
This observation was done at the end of administration period and recovery period.
During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) pectoral legs, 2) pelvis legs, 3) all four legs. Grip power was expressed in Newtons.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Time schedule: 29th day of study (main groups) and 43rd day of study (satellite groups)
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (neutral 4% formaldehyde).

HISTOPATHOLOGY: Yes
Time schedule: 29th day of study (main groups) and 43rd day of study (satellite groups)
Tissue specimens fixed in 4% neutral formaldehyde were processed by routine paraffin technique and stained by hematoxyline-eosine.
Samples of the tissues and organs were collected at necropsy and fixed. The list of examined organs is stated in the Table 5.
Other examinations:
Biometry of organs
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, thymus, spleen, brain, pituitary gland and heart were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
Statistics:
Data Processing
All the primary data (body weight, food consumption, water consumption, health condition control, general clinical observation, detailed clinical observation, functional observation, haematological examination, biochemistry, urinalysis, biometry of organs, necropsy findings, histopathological examination) were recorded in protocols. These primary data were used for calculations and processed to tables.
Statistical Analysis
The ANOVA test - Analysis of Variance (a part of software QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with satellite treated group.
The results statistically significant on probability level 0.05 were indicated by figures with asterisk in the tables of medians or averages.

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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No clinical changes were observed in animals of all dose levels after application of the test substance.
No mortality was observed in animals of all dose levels after application of the test substance.

HEALTH CONDITIONS
Males:
The health condition of animals was very good. One male treated with 250 mg/kg/day showed slight diarrhoea from 3rd to 4th week but this was incidental and unrelated to systemic toxicity. In any other groups of animals no signs of diseases or toxic effect owing to application of the test substance were found out.
Females:
The health condition of animals was very good. In any groups of animals no signs of diseases or toxic effect owing to application of the test substance were found out.

BODY WEIGHT AND WEIGHT GAIN
The body weight increment of all animals within groups was relatively well-balanced during whole application period.

FOOD CONSUMPTION
Males
Food consumption of all treated groups of males was relatively well-balanced during whole application. Slightly increased daily consumption of males in the dose level 250 mg/kg/day was recorded since the second week of application.

Females
Food consumption of animals in the dose level 250 mg/kg/day was well-balanced with the control females during whole application period. In the middle and the highest dose levels, food consumption was slightly decreased from 1st week to the last week of application period.

FOOD CONVERSION
Males
The average food conversion of treated animals was relatively well-balanced. Slightly decreased conversion was recorded in the dose levels 500 mg/kg/day in the 1st and 4th week of application period. On the contrary slightly increased conversion was recorded in the dose levels 1000 mg/kg/day at the third week of application period.
Females
The average food conversion of animals in the dose level 250 mg/kg/day was well-balanced. In the 2nd and 4th week of study, slightly increased conversion was recorded in the dose levels 500 mg/kg/day. In the dose level 1000 mg/kg/day, increase of food conversion was recorded in 2nd and 3rd week of application period.

WATER CONSUMPTION:
Males
The average water consumption of treated animals was relatively well-balanced. Slightly increased consumption was recorded in the dose levels 250 mg/kg/day in the second and third week of application period.
Females
The water consumption of all treated groups was lower against the control group during whole application period.

HAEMATOLOGY
Males
Value of APTT was increased with dose. Changes in differential leucocyte count were recorded in the highest dose level (without statistical significance). Value of granulocytes was increased and value of lymphocytes was decreased.
Other measured parameters were similar compared with animals in the control group.
All observed parameters were within physiologic limits.
Females
Statistically significant increase of Protrombin time was found in the dose levels 500 and 1000 mg/kg/day. Value of fibrinogen was slightly increased in animals of the dose level 250mg/kg/day. Increased value of granulocytes was recorded in all dose levels (without statistical significance) markedly in the middle dose level.
Other measured parameters were similar compared with animals in the control group.


CLINICAL CHEMISTRY
Males
Values of glucose increased with statistical significance in the dose level 250 and 500 mg/kg/day. Activity of ALT decreased in middle and the highest dose level (statistically significantly against control group in the dose level 1000 mg/kg/day). Slightly increased values of total protein were recorded in all dose levels. Decreased values of creatinine without statistical significance were measured in the lowest and highest dose level. In males of the highest dose level increased concentration of potassium was recorded.
Other measured parameters were similar compared with animals in the control group.
All observed parameters were within physiologic limits.
Females
Value of glucose slightly decreased with increasing dose. Statistically significantly decreased content of anorganic phosphorus was measured in the middle dose level. Slightly increased values of total protein were recorded in all dose levels. The value of creatinine was slightly increased in the dose levels 250 and 500 mg/kg/day.
Other measured parameters were similar compared with animals in the control group.
All observed parameters were within physiologic limits.

URINALYSIS
Males
Specific weight of urine was slightly decreased in the dose level 500 mg/kg/day (without statistical significance). Increased pH and slightly increased urine volume were recorded in males of the dose level 500 mg/kg/day. In this dose level, urine of three animals showed changed colour (dark). White cloud in urine was recorded in one animal of control group, in two animals of middle dose and in one animal of the highest dose level. Protein was measured in urine of one animal in the dose level 500 mg/kg/day. Leucocytes were found in control group (2 animals) and in the middle dose (1 animal).
Satellite males
Decreased of specific weight and increased urine volume were recorded in satellite treated group. Changed colour (dark) in urine of one male was recorded in the treated group. White cloud was found in urine one male of the control group. Protein and leucocytes were recorded in two animals of control group. Leucocytes were found in urine of one treated male.
Females
Specific weight of urine decreased with increasing dose (without statistical significance). Urine of one female showed changed colour (dark) in the dose levels 500 and 1000 mg/kg/day.
Satellite females
Statistically significantly decreased of specific weight was recorded in satellite treated group. Changed colour (dark) in urine was recorded in two animals of the control group. Blood in urine was recorded in one female of control group.

BEHAVIOUR
Clinical Observation
The behaviour of treated animals was similar in comparison with behaviour of animals of the control group during the whole study.
No changes were found out at all dose levels during examination of: position of eyelids, tonic movements, clonic movements, reaction to handling, lacrimation, salivation, emiction, defecation, colour of mucous membranes, vocalisation and other activity.

ORGAN WEIGHTS
Males
Absolute weight of organs was relatively well-balanced in all dose levels. Only slightly increased weight of liver were recorded in the dose levels 250 and 1000 mg/kg/day.
Females
Absolute weight of organs was relatively well-balanced in all dose levels. Only slightly increased weight of liver and uterus were recorded in the dose levels 250 mg/kg/day.

Macroscopic Findings:
Males
During the macroscopic examination of males no important pathologic changes were found. In 1-1-0-0 males no macroscopic changes were observed.
In thoracic cavity petechiae on thymus in1-0-0-0 males and focal changes on lungs in 0-1-3-0 males were diagnosed.
In abdominal cavity irregular colour or marked structure of liver in 1-1-1-2 males were found out. The macroscopic changes were more often found in stomach: mucous membrane congested in 2-1-4-4 males, focal changes of mucosa in 1-1-1-1 males, haemorrhage in 0-1-1-1 males and changed mucosa in 0-1-0-0 males.
In cranial cavity no changes were diagnosed.

Satellite males
During the macroscopic examination of males no important pathologic changes were found. In 1-1 males no macroscopic changes were observed.
In thoracic cavity focal changes on lungs in 1-2 males were diagnosed.
In abdominal cavity irregular colour or marked structure of liver in 1-1 males and marked structure of kidneys in 1-0 male were found out. The macroscopic changes were recorded in stomach: mucous membrane congested in 1-1 males, focal changes of mucosa in 1-0 male, haemorrhage in 0-1 male and changed mucosa in 2-1 males.
In cranial cavity no changes were diagnosed.
Females
During the macroscopic examination of females no important pathologic changes were found. In 1-1-1-1 females no macroscopic changes were observed.
In thoracic cavity focal changes on lungs in 1-0-0-1 females were diagnosed.
In abdominal cavity irregular colour or marked structure of liver in 2-2-2-1 females were found out. The numerous macroscopic changes were recorded in stomach: mucous membrane congested in 0-0-2-1 females, focal changes of mucosa in 1-0-0-2 females, haemorrhage in 0-0-2-0 females and changed mucosa in 0-2-1-0 females. Dilatation of uterus with fluid was diagnosed in 1-3-3-2 females.
In cranial cavity no changes were diagnosed.
Satellite females
During the macroscopic examination of females no important pathologic changes were found. In 1-1 females no macroscopic changes were observed.
In thoracic cavity focal changes on lungs in 1-2 females were diagnosed.
In abdominal cavity irregular colour or marked structure of liver in 2-0 females were found out. The numerous macroscopic changes were recorded in stomach: mucous membrane congested in 1-0 female, focal changes of mucosa in 1-1 females, haemorrhage in 0-1 female and changed mucosa in 1-1 females. Dilatation of uterus with fluid was diagnosed in 1-0 female.
In cranial cavity no changes were diagnosed.


HISTOPATHOLOGY
Histopathological Findings
By the microscopical examination of organs and tissues of laboratory rats higher incidence of the pathological findings was not found in animals in the groups administered with any of the test substance dose in comparison with the animals in control groups. Spontaneously occurred lesions were of small range.


Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption; food efficiency; water consumption and compound intake; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology;
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption; food efficiency; water consumption and compound intake; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

SUMMARY  

The test substance, Humic acids, sodium salts, was tested for subacute toxicity using the EU method B.7. Repeated Dose (28 -days) Toxicity (Oral), Directive 96/54/EC, published in OJ L 248, 1996.

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered as solution water by stomach tube; oral application of rats was made daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 5 males and 5 females; each satellite group consisted of 5 males and 5 females. Main groups contained 3 treated groups (doses 250, 500, 1000 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 (1000 mg/kg/day). The administration period lasted 28 days. After that animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.

During the 28-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 twice a week. Before the end of study the functional observation was accomplished. The study was finished by urinalysis, haematological and biochemical analysis, and gross necropsy of animals. The selected organs for weighing and histopathology examination were removed.

Results

There were no unscheduled deaths during the test. No clinically observed signs of toxicity were detected. The heath condition of animals was very good during whole study and functional observation evidenced no effect of the test substance.

No adverse effect on body weight development was detected. The body weight and body weight increment of all test groups was relatively well-balanced during whole application and recovery period. No adverse effect on dietary intake or food conversion was detected.

The haemocoagulation examination detected significant changes - prolongation of protrombine time was recorded in females of the middle and the highest dose level at the end of application period.  On the contrary the markedly decreased protrombine time were measured in both sexes after 14-day recovery period – the effect of the test substance on this blood component was protracted.  Accompanying changed value of APTT or fibrinogene were sporadically found out in both sexes. Also activities of hepatic enzyme ALT shoved incidental inter group differences.

The animals treated by the test substance showed changed parameters in clinical biochemistry and urinalysis which indicate effect on urine excretion system – increased value of protein total a creatinine, decreased concentration of anorganic phosphorus and decreased specific weight of urine.

Biometry of organs detected no significant inter group differences. No treatment-related macroscopic abnormalities or microscopic changes were detected. The morphological changes were those commonly observed in laboratory maintained rats of the strain and age employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be toxicological significance.

Overall assessment

The oral administration of Humic acids, sodium salts to rats by gavage for a period of twenty-eight consecutive days at dose level 250 and 500 mg/kg/day produced no toxicologically significant changes in the parameters measured. No major functional changes in any organ systems or severe organ dysfunction were detected. Consistent changes in clinical biochemistry, haematology and urinalysis parameters, which indicate organ dysfunction, were recorded in animals treated by test substance in the dose level 1000 mg/kg/day.

 After completion of all examinations the following effects of test substance were distinguished:

-    changes of haemocoagulation

-    negative effect on urine excretion system

Based on the results of laboratory investigations in clinical biochemistry, haematology and urinalysis it could be done the following conclusion about NOAEL:

The NOAEL (No Observed Adverse Effect Level) for MALES and FEMALES is 500 mg/kg/day.