Ashes (residues), cenospheres

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on read-across following an analogue approach:

OECD 471; GLP; S. typhimurium TA1535, TA1537, TA98, and TA100 and E.coli WP2 uvrA; 10-5000 µg/plate; not mutagenic
OECD 476; GLP; L5178Y cells; 9.77-2500 µg/ml; not clastogenic

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

09 Nov - 22 Dec 2009

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.”

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz, Freie und Hansestadt Hamburg, Germany

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells used during experimental studies were maintained in RPMI 1640 medium supplemented with 0.05% Pluronic® F68, 2 mM L-glutamine, 220 µg/mL sodium pyruvate, 100 µg/mL gentamycin, 2.5 µg/mL fungizone and horse serum (10% by volume), hereinafter referred to as growth medium. Treatment medium was growth medium without sodium pyruvate, gentamycin and fungizone. Cleansing medium used for reducing the spontaneous frequency of TK-/- mutants prior to experimental studies consists of growth medium supplemented with approximately 4.0 x 10E-5 M thymidine, 1.2 x 10E-4 M hypoxanthine, 3.3 x 10E-5 M glycine and 7.2 x 10E-7 M methotrexate. Recovery medium is similar to cleansing medium, except that the methotrexate component is removed. Selection medium is growth medium that contains 3 µg/mL of 5-trifluoro-thymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat S9 fraction

Test concentrations with justification for top dose:

9.77, 19.5, 39.1, 78.13, 156.3, 312.5, 625, 1250, 2500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

other: The vehicle served as the negative control.

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3 and 24 h without S9 mix; 3 h with S9 mix
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): 11-12 days


SELECTION AGENT (mutation assays): 5-trifluoro-thymidine (TFT) at 3 µg/mL


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A dose-related or toxicity-related increase in mutant frequency should be observed.
If the mutant frequency obtained for a single dose at or near the highest testable toxicity is ca. 4 times the concurrent background mutant frequency or greater, the test item will be considered mutagenic in a single trial. Smaller increases will require confirmation by a repeat assay.
Sometimes correlation between toxicity and applied concentration is poor. The proportion of the applied test item effectively interacting with the cells to cause genetic alterations is not always repeatable or under control. Conversely, measurable changes in frequency of induced mutants may occur with concentration changes causing only small changes in observed toxicity. Therefore, either parameter, applied concentration or toxicity, can be used to establish whether the increase in mutant frequency is related to an increase in effective treatment.
Treatments inducing less than 10% relative growth are included in the assay, but are not used as primary evidence for mutagenicity as it relates to risk assessment.
The ratio of small to large colonies will be calculated from the results of the determination of small to large colonies.
If the test item is positive, the small to large colony ratio for the test item will be compared with the corresponding ratios of the positive and negative controls. Based on this comparison the type of the mutagenic properties of the test item will be discussed.
A test item is evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 10% to 20% relative growth or in the case of relatively non-toxic items, a range or applied concentrations extending to the maximum of 5 mg/mL (or 0.01 M, or 5 µL/mL) or in the case of non-toxic, insoluble materials, a range of applied concentrations extending to at least twice the solubility limit in culture media.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to a concentration of 2500 µg/mL. The top concentration of 5000 µg/mL, as tested in the range-finding study, was a deep black coloured suspension and, thus, not analysable .

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity experiment was performed to establish an appropriate concentration range for the mutation experiment. This study was performed both with and without S9 metabolic activation.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control.
A wide range of test item concentrations of 25, 100, 250, 1000, 2500 and 5000 µg Ash/mL was tested for cytotoxicity. After an exposure time of 3 hours at approx. 37 °C on a roller drum at 10 - 15 rpm, the cells were washed and resuspended in growth medium. The cells were then adjusted to 8 cells/mL and for each dose 0.2 mL was plated into 32 microtiter wells. The plates were incubated at 37 °C in a humidified incubator gassed with 5% CO2 in air for 3 days. Wells containing viable clones were identified under a microscope and counted.
The preliminary cytotoxicity information was then used to select dose levels for the mutation assay. Doses were selected using the following criteria: At least eight analysable concentrations were used. Where there would be cytotoxicity, these concentrations covered a range from the maximum to little or no toxicity; this means that the concentration levels were separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10 - 20% (but not less than 10%) relative survival (relative cloning efficiency) or relative total growth. For relatively non-cytotoxic compounds the maximum concentration is 5 mg/mL, 5 µL/mL, or 0.01 M, whichever is the lowest. The top concentration of 5000 µg/mL, as tested in this range-finding study, was a deep black coloured suspension and, thus, not analysable .


COMPARISON WITH HISTORICAL CONTROL DATA: The mean values of mutation frequencies of the negative controls ranged from 78.84 to 83.78 per 10E6 clonable cells in the experiments without metabolic activation, and from 79.93 to 105.51 per 10E6 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

The mutation frequencies of the cultures treated with Ash ranged from 61.08 to123.73 per 10⁶clonable cells (3 hours exposure) and 80.56 to110.66 per 10⁶clonable cells (24 hours exposure) in the experiments without metabolic activation and 56.77 to111.66 per 10⁶clonable cells (3 hours exposure, first assay) and 68.53 to 97.16 per 10⁶clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.71 to 2.00 for Ash treated cells and from 0.51 to 1.36 for the negative controls.

The positive controls Methylmethanesulfonate (10 and 15 µL/mL) and 3-Methyl­cholanthrene (2.5 and 4.0 µg/mL) caused pronounced increases in the mutation frequency ranging from 4061.79 to 7547.15 per 10⁶clonable cells in the case of MMS and ranging from 3663.53 to 7380.73 per 10⁶clonable cells in the case of 3-MC.

In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.65 to 2.72 in the case of MMS.

According to the evaluation criteria for this assay, these findings indicate that a suspension of Ash, tested up to the highest analysable concentration of 2500 µg/mL in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

Conclusions:

Under the conditions of this study, a suspension of Ash, tested up to the highest analysable concentration of 2500 µg/mL in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Ash did not exhibit clastogenic potential at the concentration range investigated.
According to the evaluation criteria for this assay, these findings indicate that a suspension of Ash, tested up to the highest analysable concentration of 2500 µg/mL in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

16 Nov 2007 - 11 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.”

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium), trp operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Delor 106-induced Wistar male rats. Delor 106 is a mixture of PCBs.

Test concentrations with justification for top dose:

Toxicity test in tester strain TA 100: 10, 100, 500, 1000, 2500, 5000 µg/plate
1st and 2nd mutagenicity tests: 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubulity tests were performed prior to the main test. The test material is practically insoluble in any of the solvents commonly used and compatible with this mutagenicity test (water, dimethylsulfoxide (DMSO), dimethylformamide (DMFA), ethanol 96%, acetone, cyclohexane). DMSO was chosen due to the homogeneity of the suspensions obtained and its historical use in the testing laboratory.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.1 mL DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (20 µg/plate), 2-aminofluorene (10 µg/plate), 2-aminoanthracene (1 and 2.5 µg/plate), N-methyl-N’-nitro-N-nitrosoguanidine (20 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48-72 h


NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The main criterion for the evaluation of results was the modified two-fold increase rule. According to this rule, a result is considered positive if a reproducible dose-effect and/or a doubling of the Rt/Rc ratio is reached (Rt/Rc: ratio of the number of revertants at the tested dose to the number of revertants in the negative control).

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity as determined with tester strain TA 100 without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The effect of Ashes (residues) – Fly Ash on S. typhimurium TA 100 (two experiments).

Note: Rt/Rc: ratio of the number of revertants at the tested dose to the number of revertants in the negative control.

 

	Without metabolic activation (No. of revertants per plate)		With metabolic activation (No. of revertants per plate)
Doses (µg/plate)	Mean ± SD	Rt/Rc	Mean ± SD	Rt/Rc
Neg. control	87±7	-	108±3	-
DMSO (solvent control)	90±7	-	105±10	-
50	81±8	0.9	103±6	1.0
150	95±5	1.1	100±3	1.0
500	95±8	1.1	94±6	0.9
1500	100±8	1.1	93±2	0.9
5000	82±2	0.9	79±12	0.8
AS/2 -AF	476±16	5.3	527±22	5.0
Neg. control	125±6	-	137±4	-
DMSO (solvent control)	114±7	-	128±7	-
50	118±8	1.0	117±7	0.9
150	114±5	1.0	130±9	1.0
500	110±0	1.0	125±3	1.0
1500	108±0	1.0	129±6	1.0
5000	107±6	0.9	118±14	0.9
AS/2 -AF	560±30	4.9	998±53	7.8

 

 

The effect of Ashes (residues) – Fly Ash on S. typhimurium TA 1535 (two experiments).

Note: Rt/Rc: ratio of the number of revertants at the tested dose to the number of revertants in the negative control.

 

	Without metabolic activation (No. of revertants per plate)		With metabolic activation (No. of revertants per plate)
Doses (µg/plate)	Mean ± SD	Rt/Rc	Mean ± SD	Rt/Rc
Neg. control	23±	-	15±1	-
DMSO (solvent control)	20±13	-	18±0	-
50	24±1	1.2	17±2	0.9
150	24±2	1.2	22±2	1.2
500	23±3	1.1	16±1	0.9
1500	21±1	1.0	15±3	0.8
5000	22±3	1.1	18±2	1.0
AS/2-AA	475±22	23.3	216±17	11.8
Neg. control	25±1	-	21±2	-
DMSO (solvent control)	25±2	-	24±3	-
50	25±5	1.0	26±2	1.1
150	28±2	1.1	21±2	0.9
500	25±7	1.0	21±2	0.9
1500	24±2	1.0	17±1	0.7
5000	21±2	0.8	20±6	0.8
AS/2-AA	569±13	22.7	216±2	9.1

 

 

The effect of Ashes (residues) – Fly Ash on S. typhimurium TA 98 (two experiments)

Note: Rt/Rc: ratio of the number of revertants at the tested dose to the number of revertants in the negative control.

 

	Without metabolic activation (No. of revertants per plate)		With metabolic activation (No. of revertants per plate)
Doses (µg/plate)	Mean ± SD	Rt/Rc	Mean ± SD	Rt/Rc
Neg. control	29±4	-	32±4	-
DMSO (solvent control)	26±4	-	28±1	-
50	25±1	1.0	31±2	1.1
150	26±5	1.0	30±1	1.1
500	27±4	1.0	28±5	1.0
1500	30±3	1.2	29±9	1.0
5000	26±3	1.0	30±3	1.1
NPD/2-AF	621±14	23.9	886±71	31.3
Neg. control	37±3	-	36±2	-
DMSO (solvent control)	29±3	-	36±3	-
50	33±3	1.1	35±1	1.0
150	34±10	1.1	40±9	1.1
500	28±2	0.9	32±1	0.9
1500	31±5	1.0	34±6	0.9
5000	28±1	0.9	35±3	1.0
NPD/2-AF	984±132	33.5	1037±121	28.5

 

 

The effect of Ashes (residues) – Fly Ash on S. typhimurium TA 1537 (two experiments)

Note: Rt/Rc: ratio of the number of revertants at the tested dose to the number of revertants in the negative control.

 

	Without metabolic activation (No. of revertants per plate)		With metabolic activation (No. of revertants per plate)
Doses (µg/plate)	Mean ± SD	Rt/Rc	Mean ± SD	Rt/Rc
Neg. control	12±3	-	9±1	-
DMSO (solvent control)	13±1	-	11±3	-
50	14±2	1.0	12±1	1.1
150	9±3	0.7	14±2	1.3
500	12±2	0.9	13±4	12
1500	14±2	1.1	11±1	1.0
5000	8±0	0.6	10±2	0.9
9-AAc/2-AA	1008±30	77.5	176±25	16.5
Neg. control	18±2	-	17±2	-
DMSO (solvent control)	14±1	-	16±2	-
50	14±2	1.0	16±3	1.0
150	12±1	0.9	14±4	0.9
500	13±1	0.9	16±1	1.0
1500	13±3	0.9	15±1	1.0
5000	11±3	0.8	12±3	0.7
9-AAc/2-AA	958±123	68.4	169±20	10.8

 

 

The effect of Ashes (residues) – Fly Ash on E. coli WP2 uvrA (two experiments)

Note: Rt/Rc: ratio of the number of revertants at the tested dose to the number of revertants in the negative control.

 

	Without metabolic activation (No. of revertants per plate)		With metabolic activation (No. of revertants per plate)
Doses (µg/plate)	Mean ± SD	Rt/Rc	Mean ± SD	Rt/Rc
Neg. control	30±1	-	37±9	-
DMSO (solvent control)	30±0	-	30±6	-
50	25±1	0.8	28±5	0.9
150	28±6	0.9	27±1	0.9
500	29±1	1.0	34±2	1.1
1500	28±3	0.9	29±2	1.0
5000	32±6	1.1	32±7	1.1
MNNG/2-AA	773±43	25.8	109±7	3.6
Neg. control	26±5	-	30±5	-
DMSO (solvent control)	24±4	-	29±4	-
50	22±5	0.9	24±2	0.8
150	29±9	1.2	24±2	0.8
500	30±2	1.3	22±3	0.8
1500	30±5	1.2	24±4	0.8
5000	20±0	0.8	24±3	0.8
MNNG/2-AA	716±29	29.8	102±11	3.6

  

AS: sodium azide (1.5 µg/plate; TA 100 and TA 1535 without metabolic activation)

NPD: 4-nitro-o-phenylenediamine (20 µg/plate; TA 98 without metabolic activation)

2-AF: 2-aminofluorene (10 µg/plate; TA 100 and TA 98 with metabolic activation)

2-AA: 2-aminoanthracene (1 µg/plate with TA 1535; 2.5 µg/plate with TA 1537; 25 µg/plate with E. coli; all tests with metabolic activation)

9AAc: 9-aminoacridine hydrochloride monohydrate (100 µg/plate; TA 1537 without metabolic activation)

MNNG: N-methyl-N’nitro-N-nitrosoguanidine (20 µg/plate; E.coli without metabolic activation)

- : not evaluated

 

Conclusions:

Under the experimental conditions described, the test substance was non-mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 as well as in E. coli strain WP2 uvrA both with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on read-across following an analogue approach:

OECD 474; GLP; Wistar rat (male/female); 2000 mg/kg bw/day; not cytogenic

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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.”

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

200 instead of 500 erythrocytes for bone marrow, 2000 instead of 4000 immature erythrocytes per animal scored

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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
- 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

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

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

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

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.

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.

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

Conclusions:

Under the experimental conditions described, the test substance induced no cytogenetic damage as assessed in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are no substance specific data available on the acute toxicity of ashes (residues), cenospheres.

Ashes (residues), cenospheres and ashes (residues), coal share a common production process as substances derived from coal combustion. Ashes (residues), cenospheres represent a fraction of ashes (residues), coal separated by physical means. Both substances exhibit similarities in physicochemical properties and chemical composition. The main differences consist in a much lower content of water soluble matter and the particle size distribution of ashes (residues), cenospheres.

In terms of hazard assessment, studies available for ashes (residues), coal are therefore taken into account by read-across following an analogue approach, the results of these studies being considered a worst case for ashes (residues), cenospheres.

 

In vitro

A bacterial gene mutation assay was conducted with Ashes (residues) following a protocol compliant with OECD guideline 471 and under GLP conditions. The direct plate incorporation procedure was performed with Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 as well as with Escherichia coli WP2 uvrA at concentrations up to 5000 µg/plate in the absence and in the presence of a metabolic activator (Aroclor 1254-induced rat liver S9). No cytotoxic effects were observed up to the highest concentration as determined with S. typhimurium TA 100. Ashes (residues) did not induce mutations in the bacterial mutation tests in the absence and presence of metabolic activation in any of the strains tested. The positive and negative controls included in the experiments showed the expected results (Täublová, 2008).

Ashes (residues) were assayed in a gene mutation assay in cultured mammalian cells according to OECD guideline 476 and complying with GLP. The test material was suspended in water due to insolubility in any of the other solvents recommended. The suspension, tested up to the highest analysable concentration of 2500 µg/mL in the absence and presence of metabolic activation (S9 mix from Aroclor 1254-induced rats) in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. The positive controls included in the study exerted potent mutagenic effects. No signs of cytotoxicity (decreased survival) were noted in the experiments without and with metabolic activation, immediately after treatment (3 and 24 h without S9 mix; 3 h with S9 mix) and in the following plating for 5-trifluoro-thymidine (TFT) resistance, up to the top concentration of 2500 µg/mL. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Ashes (residues) also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that Ashes (residues), tested up to the highest analysable concentration of 2500 µg/mL in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential (LPT, 2010).

 

In vivo

The potential of Ashes (residues) to induce cytogenetic damage in vivo was investigated using Wistar rats in a GLP-study conducted according to OECD 474. The test substance was administered to the test animals by stomach tube in single dose. A dose level of 2000 mg/kg bw was chosen according to the results of a pilot experiment. Bone marrow samples were taken 24 and 48 h 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 the bone marrow were examined by light microscope. The test substance, at the dose level of 2000 mg/kg bw, did not induce a significant increase in the number of immature erythrocytes with micronuclei compared with the negative control group. It was therefore concluded that, under the experimental conditions of the study, Ashes (residues) did not give rise to the formation of micronuclei in immature erythrocytes in bone marrow of rat (Valášková, 2008).

Justification for classification or non-classification

Based on read-across following an analogue approach, the available data on the genotoxic potential of ashes (residues), cenospheres is conclusive but not sufficient for classification according to GHS (CLP, 1272/2008/EC) criteria for classification and labelling.