Ashes (residues), plant

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26.1.2009 - 5.3.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(see Overall remarks part)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues), plant

EC Number:

297-049-5

EC Name:

Ashes (residues), plant

Cas Number:

93333-79-0

Molecular formula:

Not applicable (UVCB Substance)

IUPAC Name:

Ashes (residues), plant

Details on test material:

- Name of test material: Ashes (residues) – Biomass Combustion
- Physical state: solid
- Substance type: technical product
- Appearance: black powder

- Composition of test material, percentage of components:
SiO2 37.81 % (w/w), K2O 17.01 % (w/w), CaO (total) 11.92 % (w/w), P2O5 6.15 % (w/w), Al2O3 4.05 % (w/w), SO3 (sulphate) 3.47 % (w/w), MgO 2.25 % (w/w), Fe2O3 1.81 % (w/w), CaO (free) 1.40 % (w/w), CO2 1.29 % (w/w), Na2O 0.60 % (w/w), TiO2 0.34 % (w/w).
- Impurities (identity and concentrations):
Metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn sum <
0.1%

- Lot/batch No.: BMA/F/2009
- Expiration date of the lot/batch: 15 years / 12/2024
- Storage condition of test material: PE container, temperature bellow 40°C

Method

Target gene:

gene for synthesis histidine or tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine dependent strain

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan dependent strain

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

Water for injections ( Ardeapharma, batch No. 0203200809, exp. 08/2011)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

AS

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

AAc

Migrated to IUCLID6: hydrochloride monohydrate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylenediamine (CASRN: 99-56-9)

Remarks:

NPD

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene (CASRN: 153-78-6)

Remarks:

AF

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (CASRN: 613-13-8)

Remarks:

AA

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (CASRN: 70-25-7)

Remarks:

MNNG

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3 (Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors)

DETERMINATION OF CYTOTOXICITY
- Method: total growth

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increse rule which is compatible with the aplication of staistical methods After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.

Statistics:

For the evaluation of results the modified two-fold increase rule was used, which using is comparable with application of statistical methods (2, 3).

2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240, 1980
3. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91, 1987

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Species / strain:

S. typhimurium TA 98

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

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The first mutagenicity experiments were done with the same highest dose as toxicity test. The starting dose was diluted according to guidelines (five different analysable concentrations with approximately half log (i.e. Ö10) intervals between test points). No problems occured at evaluation, so the same doses were used in the second mutagenicity experiments.

COMPARISON WITH HISTORICAL CONTROL DATA:
Spontaneous reversions, negative controls (solvent), and positive controls were compared with historical controls in VUOS laboratory. The actual numbers were in ranges of the historical numbers.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Selection of doses/toxicity
As the test substance is not soluble in any solvent, a suspension in water for injections was prepared in maximum concentration given in guidelines (5000 µg per plate). Visual inspection was performed to confirm homogeneity of the suspension.
The highest concentration was further diluted. Single test tubes were shaken before withdrawal of an aliquote for dilution as well as before withdrawal of 0.1 mL to top agar and before pouring of top agar to dishes. Concentration series arised (10-5000 µg per plate) was tested for toxicity in strain TA 100 without metabolic activation. Although the suspension of the test substance was present in top agar, the evaluation was possible without problems. No signs of toxicity were observed. In the highest concentration, a microbial contamination was observed, so that evaluation of number of revertants was not possible. No changes were observed in bacterial background.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: The effect of the test substance

S. typhimurium TA 100
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I without metabolic activation			experiment I with metabolic activation
spont. rev	0	116±13	-	30	144±14	-
H2O	0	130± 8	-	30	152± 4	-
50	0	125± 4	1.0	30	143± 4	0.9
150	0	112± 9	0.9	30	141± 5	0.9
500	0	122± 5	0.9	30	137±10	0.9
1500	0	116± 5	0.9	30	135±10	0.9
5000	0	130± 7	1.0	30	144±11	0.9
AS/2-AF	0	440± 2	3.4	20	985± 2	6.6
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	103±17	-	30	113± 6	-
H2O	0	95± 5	-	30	121± 8	-
50	0	104± 9	1.1	30	108±11	0.9
150	0	105± 6	1.1	30	97± 8	0.8
500	0	110± 2	1.2	30	103± 4	0.9
1500	0	115±10	1.2	30	125± 9	1.0
5000	0	105±13	1.1	30	117± 6	1.0
AS/2-AF	0	473±17	5.0	20	1121±112	9.3
S. typhimurium TA 1535
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I without metabolic activation			experiment I with metabolic activation
spont. rev	0	18± 3	-	30	20± 2	-
H2O	0	23± 4	-	30	17± 2	-
50	0	24± 4	1.1	30	20± 1	1.2
150	0	24± 5	1.1	30	21± 3	1.2
500	0	25± 5	1.1	30	20± 3	1.2
1500	0	21± 2	0.9	30	23± 3	1.2
5000	0	20± 3	0.9	30	23± 3	1.3
AS/2-AA	0	503± 2	22.2	20	181± 2	10.6
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	21± 1	-	30	16± 3	-
H2O	0	19± 1	-	30	16± 1	-
50	0	19± 4	1.0	30	16± 1	1.0
150	0	18± 2	0.9	30	17± 3	1.1
500	0	15± 2	0.8	30	17± 0	1.1
1500	0	20± 2	1.0	30	17± 3	1.1
5000	0	19± 2	1.0	30	17± 3	1.1
AS/2-AA	0	554±31	28.7	20	199± 9	12.7
S. typhimurium TA 98
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I with metabolic activation			experiment I with metabolic activation
spont. rev	0	25± 3	-	30	39± 1	-
H2O	0	22± 1	-	30	34± 5	-
50	0	25± 2	1.1	30	40± 6	1.2
150	0	27± 3	1.2	30	32± 0	1.0
500	0	25± 4	1.1	30	32± 3	1.0
1500	0	26± 6	1.2	30	34± 4	1.0
5000	0	22± 4	1.0	30	36± 0	1.1
NPD/2-AF	0	1446±75	64.7	20	988± 3	29.3
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	33± 3	-	30	39± 2	-
H2O	0	26± 4	-	30	38± 2	-
50	0	35± 2	1.3	30	39± 3	1.0
150	0	26± 7	1.0	30	36± 5	0.9
500	0	34± 4	1.3	30	39± 4	1.0
1500	0	30± 4	1.2	30	28± 3	0.7
5000	0	25± 4	1.0	30	31± 2	0.8
NPD/2-AF	0	1338±63	35.2	20	1595±26	42.0
S. typhimurium TA 1537
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I without metabolic activation			experiment I with metabolic activation
spont. rev	0	15± 2	-	30	15± 4	-
H2O	0	13± 2	-	30	16± 1	-
50	0	15± 1	1.1	30	18± 3	1.1
150	0	13± 4	1.0	30	21± 7	1.3
500	0	12± 1	0.9	30	17± 1	1.0
1500	0	14± 3	1.1	30	16± 3	1.0
5000	0	14± 1	1.1	30	20± 2	1.2
9-AAc/2-AA	0	1169±106	89.9	20	224±23	14.0
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	12± 1	-	30	15± 2	-
H2O	0	11± 2	-	30	14± 2	-
50	0	11± 2	1.0	30	11± 1	0.8
150	0	9± 2	0.9	30	12± 0	0.8
500	0	9± 1	0.8	30	15± 1	1.1
1500	0	10± 2	1.0	30	15± 1	1.1
5000	0	12± 3	1.1	30	11± 2	0.8
9-AAc/2-AA	0	981±72	91.9	20	120±10	8.5
E. coli WP2 uvrA
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I without metabolic activation			experiment I with metabolic activation
spont. rev	0	37± 7	-	100	38± 2	-
H2O	0	40± 2	-	100	40± 4	-
50	0	40± 7	1.0	100	34± 1	0.9
150	0	50± 6	1.3	100	37± 6	0.9
500	0	32± 1	0.8	100	43± 2	1.1
1500	0	34± 2	0.9	100	35± 2	0.9
5000	0	35± 5	0.9	100	47± 1	1.2
MNNG/2-AA	0	483±52	12.1	100	236±19	5.9
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	36± 3	-	100	33± 3	-
H2O	0	38± 3	-	100	42± 4	-
50	0	45± 6	1.2	100	43± 3	1.0
150	0	41± 8	1.1	100	41± 1	1.0
500	0	37± 3	1.0	100	41± 8	1.0
1500	0	38± 6	1.0	100	42± 3	1.0
5000	0	35± 5	0.9	100	46± 3	1.1
MNNG/2-AA	0	447±18	11.7	100	362± 9	8.7

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance Ashes (residues) - Biomass Combustion was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without as well as with metabolic activation.

Executive summary:

Test substance Ashes (residues) - Biomass Combustion was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four  indicator Salmonella typhimurium strains TA 98, TA 100,  TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in water for injections and assayed in doses of 50-5000 mg which were applied to plates in volume of 0.1 mL.

Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Ashes (residues) - Biomass Combustion was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.