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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 13, 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, potassium salts- Molecular formula: not known - UVCB substance- Molecular weight: not known - UVCB substance- Substance type: technical product- Physical state: solid- Lot/batch No.: 16. 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: 182-183 g (males), 152-153 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: 6 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:
1) Study No. 26/07/7-P: Humic acids, potassium salts - Repeated Dose (28 days) Toxicity (Oral) - Dose-range finding study.
The oral administration of test substance to rats in the dose-range finding study at the dose levels 125, 250, 500 and 1000mg/kg/day did not cause mortality and produced no dose related decrease of body weight increments, no clinical symptoms of intoxication and no significant changes of basic blood parameters.
2) Study No. 26/07/1: Humic acids, potassium salts - Acute Oral Toxicity - Acute Toxic Class Method.
Test substance administered at the dose of 2000 mg/kg caused no death of animals.
- 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 the study all experimental animals were deeply anaesthetised using diethyl ether and subsequently exsanquinated. 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:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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:
effects observed, treatment-related
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
No signs of diseases were found out during the check-in, acclimatisation, application and observation period in satellite treated females. No treatment-related effects were detected.

BODY WEIGHT AND WEIGHT GAIN
Negative effect of the test substance on growth of animals manifested in mild decrease of body weight of treated animals especially in females. This symptom had delayed character in males – in application period body weight of control and treated males were well-balanced but in postapplication period body weight of satellite treated males was decreased (statistically significantly) in comparison with satellite control. Body weight of males of the middle dose level was effected only slightly while in males of the lowest dose level body weight was not effected by application of the test substance. In females decrease of body weight was obvious at all dose level. This difference was dependent on dose level and it was irreversible – decreased body weight was also found out in treated females at the end of observation period.

FOOD CONSUMPTION
Males
At the dose levels 250 and 1000 mg/kg average food consumption was higher than in control from the 1st week to the 3rd week of study. In the last week of application period average food consumption of males of above mentioned dose levels was lower than in control. At the dose level 500 mg/kg average food consumption of males was lower compared to control for the whole time of application period. The changes were not dependent on the dose level.
Satellite males: Average food consumption of satellite treated males was lower than in satellite control males almost for the whole time of administration period (besides the 1st and the 3rd week of study).
Females
The average food consumption of treated females was nearly well-balanced with control females except the 4th week – consumption of females of the dose levels 500 and 1000 mg/kg was lower than in control females. The dose level dependence was recorded only in the 4the week.
Satellite females
The average food consumption of satellite treated females was slightly decreased in comparison with satellite control females for the whole time of application and observation period.

FOOD EFFICIENCY
Males
Average food conversion of treated males was practically similar to average food conversion of control males nearly all along the study – only in the 1st week it was decreased compared to control.
Satellite males
The average food conversion of satellite treated males was decreased in comparison with satellite control males for the whole time of application and observation period.
Females
The average food conversion of treated females was decreased compared to control for the whole time of application period – except the 3rd week at the dose level 250 mg/kg and the 1st week at the dose level 500 mg/kg. It was not dependent on the dose level.
Satellite females:
Except the 2nd and the 5th week the average food conversion of satellite treated females was lower than in satellite control females.

WATER CONSUMPTION:
Males
Average water consumption of males of the dose levels 250 and 500 mg/kg compared to control was lower in the 1st, 3rd and 4th week of application period. In males of the dose level 1000 mg/kg water consumption was increased in the 1st and the 2nd week and decreased in the 3rd and the 4th week. The dose level dependence was not detected.
Satellite males
Except the 1st week of the study the average water consumption of satellite treated males was lower than in satellite control males.
Females
The average water consumption of treated females was decreased in comparison with control females nearly for the whole time of application period – besides the dose level 500 mg/kg in the 4th week. The dose level dependence was not detected.
Satellite females
Average water consumption of satellite treated females was practically similar to average water consumption of satellite control females nearly all along the application and observation period.


HAEMATOLOGY
Haematological examination showed an effect on total leucocyte count (increase) in males of all dose levels without dose level dependence. Reversible effect on red blood component was evident in males of the highest dose level - decrease of total erythrocyte count and value of haematocrit bellow the reference control was recorded. The above mentioned alteration had no morphological counterpart in organs of haemopoietic system. At the end of observation period red blood parameters were mildly increased in satellite treated males and females. Statistically significant changes were detected only at the highest dose level – increased value of MCV in males and increased value of PT in females. Changes of value of APTT without statistical significance were detected at all dose levels: decrease in males of the highest dose level and increase in satellite treated females, males of the middle and the lowest dose level and females of the middle dose level.

CLINICAL CHEMISTRY
During biochemical examination of blood the statistically significant changes of values of liver enzymes were detected at the highest dose level and the middle dose level: increased value of ALT in satellite treated males and increased value of AST in satellite treated females. Value of ALP was higher in males of the highest and the middle dose level but without statistical significance. In females reversible hypercalcemia with dependence on dose level occured – in females of the highest and the middle dose level statistically significant increase of calcium ions was noted and the same deviation occured in females of the lowest dose level (without statistical significance). Values of none biochemical parameters exceeded the reference interval.

URINALYSIS
Statistically significantly decreased volume of urine was detected in satellite treated males and in females of the middle dose level. Lower volume of urine were accompanied with mild increase of specific gravity. By contrast in satellite treated females statistically significant increase of urine volume was observed.

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
Statistically significant differences in relative weight of organs were detected at the highest dose level: increase of relative weight of epididymides in satellite treated males and increase of relative weight of heart in satellite treated females. These differences probably cohered with decreased body weight of animals in mentioned groups.
Colouration of stomach contents by the test substance which was observed in all animals of the highest dose level during necropsy at the end of application period demonstrated slow absorption of the test substance - still 24 hours after administration the test substance was situated in application site.

GROSS PATHOLOGY
Macroscopic Findings:
Males
In 4-1-0-0 males no macroscopic changes were found out.
In digestive tract the following macroscopic affections were detected: irregular blood perfusion of stomach mucosa in 1-0-1-0 males, haemorrhages on stomach mucosa in 0-1-4-1, local changes on stomach mucosa (focal change of colour and other small foci) in 0-2-0-0 males, stomach content coloured by the test substance in 0-0-0-5 males, ochre colour of liver and marked structure of liver in 0-0-0-1 male.
Other macroscopic changes were sporadic: punctiform haemorrhages on thymus in 0-0-1-0 male, local changes on lung (small red or brown foci ) in 0-0-0-1 male, change of colour of kidney in 0-0-1-1 males, decreased testes and epididymides in 0-1-0-0 male, cavernous focus in brain of 0-0-1-0 male and congestion of pituitary gland in 0-1-0-0 male.
Satellite males
In 1–2 satellite males no macroscopic affections were recorded.
Irregular blood perfusion of mucosa in 1-1 satellite males, haemorrhages on mucosa in 1-0 satellite male, local changes on mucosa in 2-1 satellite males (focal changes of colou and blood perfussion) and tenacious film on mucosa in 0-2 satellite males were described in stomach. Ochre colour of liver was found out in 0-1 satellite male and marked structure of liver was in 1-0 satellite male. Punctiform haemorrhages were detected on thymus of 1-0 satellite male. In lung of 2-1 satellite males local changes (small foci red or brown coloured) were observed.
Females
In 3-1-1-0 females no macroscopic findings occurred.
In stomach macroscopic affections were noted relatively often: irregular blood perfusion of mucosa in 0-2-2-1 females, haemorrhages on mucosa in 0-0-2-2 females, local changes on mucosa (focal changes of colou and blood perfussion) in 0-0-1-0 females, content coloured by the test substance in 0-0-0-5 females and tenacious film on mucosa in 0-0-2-0 females. Ochre colour and marked structure of liver was detected in 1-0-0-0 female. Punctiform haemorrhages on thymus were described in 1-0-2-0 females. Dilatation of uterus (pellucid liquid) was recorded in 1-1-1-3 females.
Other changes were observed only sporadicaly.
Satellite females
All satellite females had got some macroscopic affections. The following changes occurred in stomach: haemorrhages on mucosa in 0-2 satellite females, local changes on mucosa (focal changes of colou and blood perfussion) in 1-0 satellite female and tenacious film on mucosa in 1-1 satellite females. Ochre colour of liver was observed in 1-1 satellite females and marked structure of liver was in 0-1 satellite females. In 3-3 satellite females dilatation of uterus by pellucid liquid was detected. occurrence of other macroscopic findings was sporadic.

HISTOPATHOLOGY
Histopathological Findings
Males
Microscopical affections of organs occurred very sporadicaly in control and in treated males. In 0-2-4-3 males no histological findings were detected.
In organs of digestive system only two changes were diagnosed: solitary microgranuloma in liver of 0-1-0-0 male and oedema of stomach submucosa in 0-0-0-1 male.
Haemorrhages were found out in kidneys of 0-2-0-0 males: one was in pelvic and one was in perinephric fat.
Chronic inflammation of prostate gland (lymphoid infiltration in interstitium) occurred in 0-0-0-1 male.
Satellite males
In 4-5 satellite males no histological findings were diagnosed. Only in 1-0 satellite male inflammation in prostate gland was recorded (focal lymphoid infiltration of interstitium).
Females
Microscopical findings were diagnosed in females very sporadically.
In 4-5-3-3 females no microscopical affections were noted.
In liver of 1-0-0-0 female focal inflammation was found out (sporadic lymphoid infiltration). Inflammation accompanied by desquamation of epithelium was detected in small intestine of 0-0-0-1 female. In kidney of 0-0-1-0 female haemorhage and inflammation of vessel were observed. Increase of periarteriolar lymphoid sheats was found out in 0-0-1-0 female. Dilatation of uterus lumen (hydrometra) occured in 1-0-0-1 females.
Satellite females
In 5-3 satellite females no histological effection were detected.
In 0-2 females focal erosion on stomach mucosa was diagnosed (superficial lesion on mucous membrane).



HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS

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, potassium 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.

Method

Wistar rats of SPF quality were used for testing. The test substance was administered as solution in 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.

The stability and the homogeneity of the application form in water for injections were determined before starting the main study. The concentrations of solutions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. 

Doses for the main study - 250, 500, 1000 mg/kg/day were chosen on the basis of results of the study 26/07/7P Humic acids, potassium salts – Repeated Dose (28 days) Toxicity (Oral) – Dose–range finding study.

During the 28-day study clinical observation and health status control were performed daily. The body weight and 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  

During control of food consumption, water consumption, mortality, health condition and during general clinical observation, detailed clinical observation, functional observation and histological examination of treated animals of the dose levels 1000, 500 and 250 mg/kg/day no treatment-related effects were detected.

The test substance after 28-day oral application caused  irreversible decrease of weight gains in both sexes at the highest dose level. In animals of the middle dose levels as well as at females of the lowest dose level mild decrease of body weight was recorded too. This deviation was connected with decrease of food conversion (especially in females).          

Changes in values of haematological and biochemical parameters were detected at all dose levels. But exceeding of reference interval was noted only in males of the highest dose level – decreased values of red blood component.

The urinalysis demonstrated the effect of the test substance on volume and specific gravity at all dose levels (not in males of the lowest dose level): increase of urine volume in males (except of satellite males) and decrease of urine volume and increase of specific gravity in females (except of satellite females).

Almost in all treated groups of animals (except of satellite treated males and males of the middle dose level) differences of weight of organs were noted: increase of relative weight of uterus, epididymides, heart and pituitary gland.

In all animals of main groups of the highest dose level the colouration of stomach contents by the test substance was recorded during necropsy.  

The negative effects of the test substance (decrease of body weight, decrease of food conversion, decrease of values of red blood component, changes in biochemical parameters – increase of ALT, AST and Calcium ions) were expressed especially at the highest dose level. Most of mentioned changes which were found out at the middle and at the lowest dose level had only mild intensity and they did not give evidence of adverse reaction of animal organism.          

    

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