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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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 / 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 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 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: 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:
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.