Ashes (residues), cenospheres

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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.”

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report Date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation 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

Method

Target gene:

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

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.1 mL DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (1.5 µg/plate), 4-nitro-o-phenylenediamine (20 µg/plate), 2-aminofluorene (10 µg/plate), 2-aminoanthracene (1 and 2.5 µg/plate), 9-aminoacridine hydrochloride monohydrate (100 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).

Results and discussion

Test results

Test results 1

Species / strain:

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

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

Test results 2

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions described, the test substance was nonmutagenic 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.