Ashes (residues), coal

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Sep 2009 - 23 Oct 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues), coal

EC Number:

931-322-8

Cas Number:

68131-74-8

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Ashes (residues), coal

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 98: 10, 100, 500, 1000, 2500, 5000 µg/plate
1st mutagenicity test: 50, 150, 500, 1500, 5000 µg/plate
2nd mutagenicity test: 15, 50, 150, 500, 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test material is almost insoluble in any solvent. Water was used as suspension medium.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.1 mL water

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: Slight cytotoxic effect at 5000 µg/plate (thinner bacterial background) in preliminary toxicity test in strain TA 98 without metabolic activation. The effect was not reproducible in the first series of mutagenicity testing.

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

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	149±10	-	138±2	-
Water (solvent control)	146±14	-	139±8	-
50	141±8	1.0	142±11	1.0
150	138±14	0.9	136±6	1.0
500	140±6	1.0	137±12	1.0
1500	149±9	1.0	134±16	1.0
5000	119±23	0.8	142±15	1.0
AS/2 -AF	441±13	3.0	1235±41	8.9
Neg. control	132±7	-	134±9	-
Water (solvent control)	142±8	-	138±8	-
15	133±4	0.9	136±17	1.0
50	133±5	0.9	121±12	0.9
150	128±4	0.9	121±5	0.9
500	132±11	0.9	120±6	0.9
1500	131±4	0.9	96±14	0.7
AS/2 -AF	489±3	3.5	1132±49	8.2

 

 

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	29±2	-	19±3	-
Water (solvent control)	27±1	-	21±2	-
50	26±5	1.0	17±1	0.8
150	27±1	1.0	18±3	0.9
500	25±1	0.9	22±3	1.0
1500	30±4	1.1	18±2	0.9
5000	25±6	0.9	21±1	1.0
AS/2-AA	431±5	15.9	203±13	9.6
Neg. control	25±4	-	15±3	-
Water (solvent control)	24±4	-	16±3	-
15	26±2	1.1	14±4	0.9
50	22±4	0.9	17±6	1.1
150	24±3	1.0	17±2	1.0
500	25±5	1.1	16±3	1.0
1500	22±3	0.9	17±1	1.0
AS/2-AA	567±14	24	228±21	14.2

 

 

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	31±2	-	28±2	-
Water (solvent control)	27±4	-	25±3	-
50	25±1	0.9	29±6	1.2
150	35±8	1.3	25±4	1.0
500	23±2	0.9	29±4	1.2
1500	28±4	1.0	29±6	1.2
5000	25±5	0.9	26±5	1.1
NPD/2-AF	542±18	20.1	1261±64	51.1
Neg. control	30±5	-	34±4	-
Water (solvent control)	29±4	-	35±5	-
15	30±3	1.0	33±1	0.9
50	32±4	1.1	40±3	1.1
150	28±4	1.0	47±6	1.3
500	36±7	1.2	37±6	1.0
1500	31±1	1.0	32±5	0.9
NPD/2-AF	818±92	27.9	1722±67	48.7

 

 

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	13±3	-	12±1	-
Water (solvent control)	12±2	-	14±2	-
50	11±0	0.9	14±1	1.0
150	14±2	1.2	11±1	0.8
500	14±2	1.1	21±3	1.5
1500	11±1	0.9	14±2	1.0
5000	11±1	0.9	14±0	1.0
9-AAc/2-AA	1184±7	98.6	160±12	11.1
Neg. control	11±3	-	15±2	-
Water (solvent control)	11±2	-	16±2	-
15	15±5	1.3	17±2	1.0
50	12±1	1.1	17±2	1.0
150	13±1	1.2	15±1	0.9
500	14±1	1.2	15±2	0.9
1500	12±1	1.1	17±3	1.1
9-AAc/2-AA	1077±48	97.9	171±16	10.5

 

 

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	28±3	-	32±4	-
Water (solvent control)	24±3	-	35±2	-
50	24±2	1.0	26±5	0.7
150	25±5	1.0	29±4	0.8
500	28±4	1.2	27±2	0.8
1500	26±2	1.1	36±2	1.0
5000	31±4	1.3	18±2	0.5
MNNG/2-AA	534±17	22.2	140±20	3.9
Neg. control	32±4	-	42±2	-
Water (solvent control)	40±3	-	35±5	-
15	39±8	1.0	45±3	1.3
50	33±6	0.8	40±3	1.1
150	41±5	1.0	39±7	1.1
500	32±4	0.8	39±0	1.1
1500	37±2	0.9	41±9	1.2
MNNG/2-AA	581±16	14.5	325±16	9.2

  

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:

Under the experimental conditions described, the test substance Ashes (residues) - Fly Ash 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.