Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
27 Nov 2007 - 30 Jan 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.”

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
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): Ashes (residues)
- Substance type: technical product
- Physical state: solid
- Significant impurities:
Cr, Cu, Zn - metals with content lower than 0.1 %
As, Pb, Cd, Hg - metals with content lower than 0.01 %
- Composition of test material, percentage of components:
SiO2 (42.12 %), Al2O3 (32.16 %) and Fe2O3 (10.88 %) – main components
TiO2, MnO, MgO, CaO, Na2O, K2O, SO3 – components with content lower than 10 %
- Lot/batch No.: 30.08.2007_ENERGETIKA
- Expiration date of the lot/batch: unlimited
- Stability under test conditions: stable
- Storage condition of test material: labotatory conditions
- Appearance: greyish black powder

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, 28125, Czech Republic, RČH CZ 21760152
- Age at study initiation: 6-7 weeks
- Weight at study initiation: females 169.60 - 177.62 g, males 200.81 - 212.09 g
- Assigned to test groups randomly: yes
- Diet (e.g. ad libitum): Complete pelleted diet for rats ad libitum (ST 1 Bergman, Mill Kocanda, Jesenice by Prague)
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%):30-70
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 27 Nov 2007 To: 07 Dec 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% methylcelulose in water
- Concentration of test material in vehicle: adjusted to body weight
- Amount of vehicle (if gavage or dermal): 1 mL/100 g bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in 0.5% methylcelulose in water prior to administration via oral gavage, the concentration in vehicle was adjusted to body weight in order to achieve a constant administration volume of 1 mL/100 g bw.
Duration of treatment / exposure:
24 and 48 h
Frequency of treatment:
Single administration
Post exposure period:
Not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
30
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide monohydrate
- Route of administration: intraperitoneal injection (solution in water)
- Doses / concentrations: 20 mg/kg bw . The dose level of cyclophosphamide was chosen on the basis of literature data.
- Duration of exposure: 24 h
- No. of animals per sex per dose: 5

Examinations

Tissues and cell types examined:
Bone marrow cells from the femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The appropriate concentrations of the test item were determined from a pilot experiment.

DETAILS OF SLIDE PREPARATION:
Bone marrow harvest
Bone marrow cells were obtained from the femora immediately after animal sacrifice. After excising and careful cleaning of the bone, both femur ends were clipped off. The bone marrow was gently flushed from the bone with 1 mL of bovine serum into a tube. The acquired bone marrow was suspended and mixed in the bovine serum using a syringe with thin needle.

Preparation of bone marrow smears
After centrifugation (5 min, 1000 rpm), the supernatant was gently removed. One drop of bovine serum was added to the cell sediment which was then resuspended by mixing. One drop of the cell suspension was placed onto a slide and a smear was prepared using a pusher slide. Two bone marrow smears were prepared per animal.

Staining of the bone marrow smears
After drying (20 minutes, 60 °C) the smears were fixed with ethanol for 5 min, rinsed twice with distilled water and stained with 5% Giemsa solution for 15 min.

Examination of the bone marrow smears
Stained smears were examined with a light microscope. 200 erythrocytes were evaluated per animal as to 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
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.
Statistics:
The proportion of immature erythrocytes (cytotoxic index) and the number of micronucleated immature erythrocytes were determiend for each animal. The final count of micronucleated immature erythrocytes was adjusted to counts per 2000 erythrocytes per animal.
Mean values and standard deviations were calculated for each group of animals.
The statistical analysis was performed by the Analysis of Variance (ANOVA) test (software QC.Expert 2.5, Trilobyte, Statistical Software, Czech Rep.). Each of treatment groups was compared to the negative control group.

Results and discussion

Test results
Sex:
male/female
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
Pilot experiment:
The experiment was performed because there were no suitable data, which could be used for the dose selection. The pilot test was performed using the same strain of animals and the same treatment regimen used in the main study.
The test substance was administered by gavage using a stomach tube. Clinical symptoms of toxicity were monitored after application. After euthanasia the indication of cytotoxicity in the bone marrow was evaluated by the ratio of immature erythrocytes to the total number of erythrocytes (“cytotoxicity index”). This ratio was determined from 200 erythrocytes.

Dose levels in pilot experiment and duration of exposition:
In the pilot experiment, 15 females (12 in the treatment groups and 3 animals in the control group) were used.

Negative control and three dose levels of the test substance were tested in the pilot experiment:
1. 2000 mg/kg bw for 24 h (3 animals)
2. 2000 mg/kg bw for 48 h ( 3 animals)
3. 1000 mg/kg of bw for 24 h (3 animals)
4. 500 mg/kg bw for 24 h (3 animals)
5. negative control group: 0.5% methylcelulose in water (3 animals)

Clinical observations:
No symptoms of toxicity were observed at any dose level.

Bone marrow harvest:
For the determination of the “cytotoxicity index”, bone marrow was harvested from all animals at 24 h after application and at the dose level 2000 mg/kg bw also at 48 h after application.

Evaluation of the pilot experiment and selection of doses:
A decrease in the “cytotoxicity index” was not observed at any dose level.
For the main study the dose level 2000 mg/kg bw was chosen (limit test).


RESULTS OF DEFINITIVE STUDY
Clinical observations:
No animal died during the main experiment and no signs of toxicity were observed in any animal in the treatment groups as well as in the negative and positive control groups.

Examination of the bone marrow smears:
The ratio of immature erythrocytes to total erythrocytes is shown in Table 1.

No statistically significant changes in the ratio of immature erythrocytes to the total number of erythrocytes were observed. A statistically significant decrease was seen in the positive control group compared to the negative control.

Count of micronucleated immature erythrocytes:

The mean numbers of micronucleated immature erythrocytes (IME) in males and females are summarised in the tables 2 and 3, respectively.
No statistically significant change in the number of micronucleated immature erythrocytes were observed. A statistically significant increase in the number of micronucleated immature erythrocytes was observed in the positive control group compared to the 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

0.378

0.05

0.398

0.05

Negative control 48 h

0.380

0.05

0.375

0.05

2000 mg/kg

24h

0.377

0.02

0.369

0.04

2000 mg/kg

48h

0.386

0.05

0.399

0.06

Positive control

24h

 0.249*

0.02

0.225*

0.03

Without application

0.361

0.04

0.378

0.02

Table 2.   Micronucleated immature erythrocytes - group means and standarddeviations

MALES

Number of micronucleated IME per animal (per 2000 IME)

Percentage expression

      Group

Mean

Standard deviation

Mean

Standard deviation

Negative control 24 h

2.77

0.83

0.139

0.04

Negative control 48 h

2.58

0.54

0.129

0.03

2000 mg/kg

24h

2.58

0.55

0.129

0.03

2000 mg/kg

48h

2.77

0.83

0.138

0.04

Positive control

24 h

15.73*

1.60

0.786

0.08

Without application

2.17

0.82

0.109

0.04

 

 

Table 3.   Micronucleated immature erythrocytes - group means and standard deviations

FEMALES

Number of micronucleated IME per animal (per 2000 IME)

Percentage expression

       Group

Mean

Standard deviation

    Mean

Standard deviation

Negative control 24 h

2.38

0.88

0.119

0.04

Negative control 48 h

2.19

0.80

0.108

0.04

2000 mg/kg

24h

2.16

0.80

0.108

0.04

2000 mg/kg

48h

2.78

1.08

0.139

0.05

Positive control

24 h

18.50*

2.07

0.925

0.10

Without application

2.55

0.52

0.128

0.03

IME – immature erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions described, the test substance induced no cytogenetic damage as assessed in the micronucleus test.