Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from August 21, 2007 to September 17, 2007
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:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes

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:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeding farm BioTest s.r.o., Konarovice, 281 25, Czech Republic,
- Age at study initiation: 10 - 12 weeks at the time application
- Weight at study initiation: 215 - 242 g
- Fasting period before study: 12 hours
- Housing: animal room with monitoring conditions – 3 animals of one sex in one plastic breeding cage Velaz T4
- Diet (e.g. ad libitum): ST 1 BERGMAN – standard pelleted diet ad libitum, (producer: Mill Kocanda, Jesenice u Prahy)
- Water (e.g. ad libitum): drinking tap water ad libitum (quality corresponding to Regulation No. 252/2004 Czech Coll. of Law)
- Acclimation period: 26 days
- Randomisation: according to the internal rule, at the start of the study the weight variation of animals was minimal and did not exceed + 20 % of the mean weight
- Identification of animals: colour marks 1 - 3 on tail of animals, each cage was marked with the number of study, sex and dose of the test substance
- Health condition: certificate of good health condition – from breeding farm; no signs of diseases were observed at clinical check-in, during the acclimatisation period and before the start of study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature 22 +/- 3°C, permanently monitored
- Humidity (%): relative humidity 30 – 70 %, permanently monitored
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle:
Preparation and application of the test substance
Immediately before application the test substance was weighed, mixed in vehicle (vehicle Aqua for injectione) and resulting suspension was administered to the stomach by tube. The single volume of administered suspension was 2ml/100 g of animal body weight

- Justification for choice of vehicle: solubility
- Purity:
Aqua pro injectione (water for injections)
Bieffe Medital S.p.A. Italy
sterile, pellucid liquid without taste, colour and smell

MAXIMUM DOSE VOLUME APPLIED: 2ml/100 g of animal body weight

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: starting dose 2000 mg/L was selected as limit test
Doses:
2000 mg/kg of animal body weight
No. of animals per sex per dose:
6 animals-females
Step No. 1: 3 females
Step No. 2: 3 females

Control animals:
no
Details on study design:
Testing schedule (according to EU Method B.1 tris Annex 1D)
START: 2000 mg/kg – 3 females (Step No.1): no deaths ► 2000 mg/kg – 3 females (Step No. 2): no deaths ► END of study

- Duration of observation period following administration: 15 days
- Frequency of observations and weighing: Animals were weighed before application, on the 8th day of study and at day 15, before euthanasia of animals.
After application the animals were observed individually – the first day: twice (30 minutes and 3 hours after application), the second day: twice (in the morning and in the afternoon) and daily thereafter for 14 days.
Observations included changes in skin and fur, eyes, visible mucous membranes, behaviour of animals, somatomotor activity, reactions to stimuli, presence of lacrimation, salivation and discharge from nostrils, function of respiratory, digestive and urogenital system.

- Necropsy of survivors performed: yes
All test animals survived to the end of study were sacrificed on the 15th day by injection of veterinary preparation T61 (1 ml iv.) and gross necropsy was carried out. Nutritious status, body surface, body foramina, thoracic, abdominal and cranial cavity were evaluated

Results and discussion

Preliminary study:
no
Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
no mortality
Clinical signs:
At the dose level of 2000 mg/kg no animal died during the study.
30 minutes after application clinical signs of intoxication were detected in all animals.
3 hours no clinical signs of intoxication were detected in all animals.
In the next morning no clinical signs of intoxication were detected in all animals.

Body weight:
Weight increments were adequate to species, sex and age of animals in experiment
Gross pathology:
All test animals survived to the end of study were sacrificed on the 15th day by injection of veterinary preparation T61 (1 ml iv.) and gross necropsy was carried out. Nutritious status, body surface, body foramina, thoracic, abdominal and cranial cavity were evaluated.
Without pathological changes.

Any other information on results incl. tables

Clinical observation - 2000 mg/kg (Step No.1)

Animal

No.

Death after

application

Observed changes

1

-

30 minutes: piloerection

3 hours: no clinical signs of intoxication

2nd – 14th day: no clinical signs of intoxication

2

-

30 minutes: piloerection

3 hours: no clinical signs of intoxication

2nd – 14th day: no clinical signs of intoxication

3  

-

30 minutes: piloerection

3 hours: no clinical signs of intoxication 

2nd – 14th day: no clinical signs of intoxication

Clinical observation - 2000 mg/kg (Step No.2)

Animal

  No.

Death after

application

Observed changes

4

-

30 minutes: piloerection

3 hours: no clinical signs of intoxication

2nd – 14th day: no clinical signs of intoxication

5

-

30 minutes: piloerection

3 hours: no clinical signs of intoxication

2nd – 14th day: no clinical signs of intoxication

6

-

30 minutes: piloerection

3 hours: no clinical signs of intoxication

2nd – 14th day: no clinical signs of intoxication

Applicant's summary and conclusion

Interpretation of results:
practically nontoxic
Remarks:
Migrated information Criteria used for interpretation of results: EU
Executive summary:

The aim of the study was to investigate acute toxic effects of the test substance Humic acids, sodium salts after a single oral administration to Wistar rats.

The testing was performed according to the Method B.1 tris: Acute Oral Toxicity - Acute Toxic Class Method, Directive 2004/73/EC, published in O.J. L152, 2004.

Test substance was administered in a single dose as a solution in Aqua pro injectione (vehicle), given orally via gavage to two groups of three female Wistar rats.

The dosing was performed sequentially in two groups of three females: group No. 1 - first step and group No.2 - second step using the starting dose of 2000 mg/kg body weight.

The test substance administered at the dose of 2000 mg/kg caused no death of animals. Clinical signs of intoxication were observed near all six animals (piloerection). Macroscopic changes not were diagnosed during pathological examination near all animals in both groups.

According to the study results the value of LD50of the test substance for female rats is higher than 2000 mg/kg of body weight.