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Toxicological information

Genetic toxicity: in vitro

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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

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 / strainopen allclose all
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 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 resultsopen allclose all
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
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.