Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from January 16, 2008 to April 16, 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:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

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: Breeding farm BioTest s.r.o., Konárovice, 281 25 Czech Republic
- Age at study initiation: 6 – 7 weeks
- Weight at study initiation: males - cca 208-210 g
females - cca 173 - 175 g
- Fasting period before study: no
- Housing: Sterilized shavings of soft wood, Monitored conditions, microbiologically defined background
- Diet: ad libitum: Complete peleted diet for rats ad libitum (ST 1 Bergman)
- Water: ad libitum, Drinking tap water
- Acclimation period: 5 days
- Stock and health condition: No signs of disease should be observed at clinical check-in.
- Prophylactic arrangement:Cleaning and disinfection of animal room
- Total number of animals:
Pilot experiment: 9 animals for determination of toxicity of the test substance (females, single administration)
Main test: 60 animals (30 males and 30 females)
- Identification of animals: Individual labelling of cages and labelling of the animals

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s) used: Water for injection - Aqua pro injectione
Manufacturer: Ardeapharma Ševětín
- Justification for choice of solvent/vehicle: solubility and relative stability of test substance in water
- Amount of vehicle: Volume of the application form was constant at all dose levels - 1 ml/100g b.w. by adequate adjusting the
concentration.
- Lot/batch no.: 0306260707
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dissolution of test substance in water for injections in appropriate amounts

Duration of treatment / exposure:
1) 2000 mg/kg, exposition – 24 h: 5 males + 5 females
2) 2000 mg/kg, exposition – 48 h: 5 males + 5 females
Frequency of treatment:
The test substance was administered to animals by stomach tube in single dose.
Post exposure period:
24 and 48 hours
During the experimental part of study, clinical observation of animals was performed every day. In all animals possible clinical symptoms of intoxication and health condition were examined 1 hour after application and in the afternoon after application.

Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg b.w.
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate
Manufacturer: SIGMA – ALDRICH CHEMIE GmbH
Batch number: 091K1176
CAS No.: 6055-19-2

- Justification for choice of positive control(s): Positive control substance was chosen according to the methodology
- Route of administration: intraperitoneal injection
- Doses / concentrations: 20 mg/kg, exposition-24h: 5 males+ 5 females

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Doses for main test were selected from results of pilot experiment.
Dose level 2000 mg/kg b.w. was chosen.

DETAILS OF SLIDE PREPARATION:
Bone marrow harvesting
Bone marrow cells have been obtained from the femora immediately after the sacrifice of animal.
After excising and careful cleansing of the bone both femur ends were be clipped off. Marrow was be gently flushed from the bone by 1 ml of bovine serum into the tube. Acquired bone marrow was several times mixed by syringe with thin needle.
Preparation of the bone marrow smears
The bone marrow with serum in tubes was centrifuged (5 min – 1000 rpm). The supernatant was gently removed, one drop of bovine serum was
added to the sediment and this cell suspension is mixed on mixer.
Clean and degreased slides were marked by the number of study, number of animal, sex and dose level. One drop of cell suspension was placed onto the slide and a smear was prepared using a pusher slide. Two slides were prepared per animal.
Staining of the bone marrow smears
After drying (20 minutes, 60°C) the smears were fixed by ethanol – 5 minutes. Then they were twice rinsed by distilled water and stained by 5% solution of Giemsa – 15 minutes.

METHOD OF ANALYSIS:
Examination of the bone marrow smears
Before examination, the slides were coded.
Stained smears were examined by light microscope. 200 erythrocytes were evaluated per animal for the proportion of immature (polychromatic) and mature (normochromatic) erythrocytes („cytotoxicity index“) in bone marrow.
At least 2000 polychromatic erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.
Criteria for distinguishing the micronuclei
colour – purple
form – generally round or almond shaped, (occasional lightly stained and ring shaped micronuclei may occur)
borders – sharp
size – 5-20% of polychromatic erythrocyte size
Scoring of micronucleated immature erythrocytes
- number of immature erythrocytes with micronuclei
(number of micronuclei per PE is not a result, occasional more than one micronucleus may appear per PE).

Data treatment
Individual animal data were summarized to tables. Proportion of immature erythrocytes (cytotoxicity index) and count of micronucleated immature erythrocytes were determined for each animal. Because the count of evaluated erythrocytes was not the same in each animal (but at least 2000 erythrocytes were evaluated), the final count of micronucleated immature erythrocytes was adjusted for 2000 erythrocytes per animal.
The Excel software was used for calculation of mean values and standard deviations for each group of animals.
The statistical analysis was performed by the ANOVA test - Analysis of Variance (software QC.Expert 2.5, Trilobyte, Statistical Software, Czech Rep.). Each of treatment groups was confronted with negative control group


Evaluation criteria:
The criteria for determining a positive result are dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. Biological relevance of the results is considered first. Statistical method (ANOVA) is used as an aid in evaluating the test results. A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test. Equivocal results could be clarified by further testing preferably using a modification of experimental conditions.
Positive results in the micronucleus test indicate that the substance induces micronuclei, which are the result of chromosomal damage or damage of the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce micronuclei in the immature erythrocytes of the test species.
A test substance for which the results do not meet the above criteria for positive result is considered non-mutagenic in this test.

Statistics:
ANOVA is used as an aid in evaluating the test results

Results and discussion

Test resultsopen allclose all
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg b.w.
- Clinical signs of toxicity in test animals: no
- Evidence of cytotoxicity in tissue analyzed: no
- Harvest times: 24 and 48 hours after application

RESULTS OF DEFINITIVE STUDY
Proportion of immature erythrocytes among total erythrocytes – “cytotoxicity index”
In group of animals administered by the substance statistically significant changes of proportion of immature erythrocytes from total number of erythrocytes were not found out. In group of animals administered by positive control, statistically significant change of proportion of immature erythrocytes from total number of erythrocytes was found out (in comparison with negative control). - see Table 1

Count of micronucleated immature erythrocytes
In Tables (2 and 3) the group means and standard deviations of micronucleated immature erythrocytes (IME) counts are summarized.
In group of animals administered by the test substance, statistically significant changes of count of micronucleated immature erythrocytes were not found out.
In group of animals administered by positive control, statistically significant change of count of micronucleated immature erythrocytes was found out (in comparison with negative control).



Any other information on results incl. tables

Table 1: Cytotoxicity index  - group means and standard deviations

MALES

FEMALES

Group

Mean

Standard deviation

Mean

Standard deviation

Negative control

- 24 h

0.444

0.04

0.426

0.02

Negative control

- 48 h

0.396

0.05

0.418

0.06

2000 mg/kg

- 24h

0.419

0.03

0.409

0.06

2000 mg/kg

- 48h

0.412

0.04

0.412

0.05

Positive control

- 24h

 0.297*

0.02

0.308*

0.02

Without application

0.419

0.08

0.417

0.06

Table 2:   Micronucleated immature erythrocytes - group means and standard deviations (males)

MALES

Number of micronucleated IME per animal (per 2000 IME)

Percentage expression

       Group

Mean

Standard deviation

Mean

Standard deviation

Negative control - 24 h

2.76

0.82

0.138

0.04

Negative control - 48 h

2.58

0.54

0.129

0.03

2000 mg/kg

- 24h

2.54

0.82

0.127

0.04

2000 mg/kg

- 48h

2.98

0.71

0.149

0.04

Positive control - 24 h

18.30*

2.60

0.915

0.13

Without application

3.19

0.84

0.159

0.04

Table 3: Micronucleated immature erythrocytes - group means and standard deviations (females)

FEMALES

Number of micronucleated IME per animal (per 2000 IME)

Percentage expression

        Group

Mean

Standard deviation

     Mean

Standard deviation

Negative control

- 24 h

2.74

0.77

0.119

0.04

Negative control

- 48 h

2.38

1.13

0.119

0.06

2000 mg/kg

- 24h

2.38

1.13

0.119

0.06

2000 mg/kg

- 48h

2.57

0.53

0.128

0.03

Positive control

- 24 h

18.33*

2.30

0.916

0.11

Without application

2.59

0.89

0.129

0.04

IME – immature erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

The test substance was tested for the assessment of cytogenetic damage in vivo, using laboratory rat (Wistar).

The test was performed according to the EU Method B.12, Mutagenicity – In vivo Mammalian Erythrocyte Micronucleus Test. The method is analogous to the OECD Test Guideline No. 474, Mammalian Erythrocyte Micronucleus Test.

The test substance was administered to animals by stomach tube in single dose. One dose level was chosen according to the results of pilot experiment - 2000 mg/kg of body weight. Two bone marrow sampling intervals were used - 24 and 48 hours after administration. The group of animals without administration and concurrent negative and positive controls were included. Each experimental group consisted of five males and five females.     

The smears obtained from bone marrow were examined by light microscope.

The test substance at the dose level 2000 mg/kg did not cause a significant increase of count of immature erythrocytes with micronuclei in comparison with negative control group.

           

At given experimental conditions the test substance, Humic acids, sodium salts, did not give rise to formation of micronuclei in immature erythrocytes in bone marrow of rat.