Ashes (residues), plant

Genetic toxicity: in vivo

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:

27.11.2007-30.1.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The 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

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues)

EC Number:

268-627-4

EC Name:

Ashes (residues)

Cas Number:

68131-74-8

Molecular formula:

Not available / UVCB substance

IUPAC Name:

FBC Ash (residues of coal combustion in fluidized bed combustion boilers)

Details on test material:

- Name of test material (as cited in study report): Ashes (residues)
- Molecular formula (if other than submission substance): not available
- Molecular weight (if other than submission substance): not available
- Smiles notation (if other than submission substance): not available
- InChl (if other than submission substance): not available
- Structural formula attached as image file (if other than submission substance): not available
- 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 or test system 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
- Random selection: according to SOP No.42
- Total number of animals: Pilot experiment: 15 animals for determination of toxicity of the test substance (single administration, acute toxicity test)Main test: 60 animals
- Identification of animals: Individual labelling of cages and labelling of the animals
- Fasting period before study: no
- Housing: Sterilized shavings of soft wood
- Diet (e.g. ad libitum): Complete peleted 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+/-3oC, permanently monitored
- Humidity (%): 30-70%, permanently monitored
- Air changes (per hr): once per hr
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am


STUDY TIME SCHEDULE:
Pilot experiment: 23. 10. – 26. 11. 2007
Main study: 27. 11. – 7. 12. 2007

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% methylcelulose (in water for injections)
Methylcelulose
Manufacturer: Dr. Kulich Pharma s.r.o.
Piletická 178/61, Hradec Králové, 500 03
Batch No.: DT157078

Water for injections
Manufacturer:Ardeapharma Ševětín a.s.
Batch No.: 0306260707


- Application of the test substance: The volume of the application form at all dose levels and negative control was constant - 1 ml/100 g of body weight by adequate adjusting the concentration. The animals were weighed before application. The application form of the test substance was prepared immediately before administration.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance was administered in 0.5% methylcelulose (in water for injections) by gavage using a stomach tube.

Duration of treatment / exposure:

The appropriate concentrations of the test item determined from pilot experiment were as follows:
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 in single dose.

Post exposure period:

24 hrs, 48 hrs

Doses / concentrations

Remarks:

Doses / Concentrations:
2000 mg/kg b.w.
Basis:
nominal conc.
in 0.5% methylcelulose (in water for injections)

No. of animals per sex per dose:

60 animals (30 males and 30 females) were used in the main study.

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide monohydrate
- Batch No. 091K1176
- Route of administration: Positive control was administered by intraperitoneal injection (solution in water for injection).
- Doses / concentrations: cyclophosphamide 20 mg/kg, time interval – 24 h: 5 males + 5 females. A dose level for cyclophosphamide was chosen on the base of literature data.

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 pilot experiment.

DETAILS OF SLIDE PREPARATION:
Bone marrow harvesting
Bone marrow cells were obtained from the femora immediately after euthanasia of animals. After excising and careful cleaning of the bone both femur ends were clipped off. Marrow was 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 was 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 bone marrow smears 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.

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, occasionally more than one micronucleus may appear per PE).

DATA TREATMENT:
Individual animal data were summarized to tables in Final Report. 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.

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 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.

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. Pilot test was performed using the same strain of animals and the same treatment regimen that was used in the main study.
The test substance was administered by gavage using a stomach tube. Clinical symptoms of toxicity were observed after application. After euthanasia the indication of cytotoxicity in the bone marrow was evaluated by the proportion of immature erythrocytes among total erythrocytes (in the further text it is referred as “cytotoxicity index”). This proportion was counted from 200 erythrocytes.

Dose levels in pilot experiment and duration of exposition
In a pilot experiment a number of 15 animals – 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 of body weight – time interval – 24 h: 3 animals
2. 2000 mg/kg of body weight – time interval – 48 h: 3 animals
3. 1000 mg/kg of body weight – time interval – 24 h: 3 animals
4. 500 mg/kg of body weight – time interval – 24 h: 3 animals
5. negative control 0.5% methylcelulose (in water for injections) – 3 animals

Clinical observation
No symptoms of toxicity were observed in animals of the dose levels 500, 1000 and 2000 mg/kg.
No symptoms of toxicity were observed in animals of the negative control group of animals.

Bone marrow harvesting
For the determination of the “cytotoxicity index”, bone marrow was harvested from all animals at 24 hours after application and at the dose level 2000 mg/kg of body weight also at 48 hours after application of the test substance.

Evaluation of the pilot experiment - selection of doses
Decrease of “cytotoxicity index” was not recorded at any of dose levels.
For the main study the dose level 2000 mg/ kg of body weight was chosen (limit test), so a full study using three dose levels was not necessary to perform.


RESULTS OF DEFINITIVE STUDY
Clinical observation
No animal died during the main experiment.
No symptoms of toxicity were observed in all animals of the dose level 2000 mg/kg.
No symptoms of toxicity were observed in the animals of positive control group and negative control groups.

Examination of the bone marrow smears
Proportion of immature erythrocytes among total erythrocytes – “cytotoxicity index” (Table 3)

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).
Individual counts of immature and mature erythrocytes from 200 erythrocytes are shown in tables 12 – 23 in Annex in Final Report.


Count of micronucleated immature erythrocytes (Tables 4 and 5)

In the tables (4 and 5) 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).
Individual counts and percentage expression of micronucleated immature erythrocytes from 2000 immature erythrocytes are presented in tables 12 – 23 in Annex in Final Report.

DISCUSSION OF THE RESULTS

At group of animals treated by the test substance at the dose level 2000 mg/kg the decrease of the proportion of polychromatic erythrocytes was not so substantial to obstruct the analysis of slides for the incidence of micronuclei. At no animal the count of polychromatic erythrocytes was less than 20% of the control value (limit value stated in the guideline).

Comparison of values between treated group and control group revealed that the test substance did not significantly affect the formation of new erythrocytes.

The count of polychromatic erythrocytes with micronuclei after 24-hour and 48-hour exposition was not changed in treated animals.

In the positive control group (with cyclophosphamide) the cytotoxicity index and count of polychromatic erythrocytes with micronuclei were statistically significantly different from negative control counts. This result demonstrates, that method performed in conditions of our laboratory has enough sensitivity to detect the endpoint in view.

Any other information on results incl. tables

Table 3.   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 4.   Micronucleated immature erythrocytes - group means and standard deviations

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 5.   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
The test substance Ashes (residues), was tested for the assessment of cytogenetic damage using micronucleus test.

Statistically significant increase in the count of micronucleated immature erythrocytes compared to the control was not recorded at any dose level.

At given experimental conditions the test substance, Ashes (residues), did not give rise to formation of micronuclei in immature erythrocytes in bone marrow of rat.

Under the given test conditions, the test substance, Ashes (residues), had negative result in micronucleus test what means that it is considered non-mutagenic in this test.

Executive summary:

The test substance, Ashes (residues), was tested for the assessment of cytogenetic damage in vivo, using laboratory rat (Wistar). The study is a part of the test substance health hazard evaluation.

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, Ashes (residues), did not give rise to formation of micronuclei in immature erythrocytes in bone marrow of rat.

 

Under the given test conditions,the test substance, Ashes (residues), had negative result in micronucleus test what means that it is considered non-mutagenic in this test.