Dust, steelmaking

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:

8.12.2009-1.2.2010

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:

In case of the strain TA 98, frozen stock culture after expiration of minimum shelf life (two years) was used for experiments - a deviation from SOP. The bacteria were tested for their properties (fenotype confirmation, response to positive controls). Al

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dust, steelmaking

EC Number:

266-005-7

EC Name:

Dust, steelmaking

Cas Number:

65996-72-7

IUPAC Name:

Dust steelmaking

Details on test material:

- Physical state: solid
- Composition of test material, percentage of components: Fe total 57.64% (mainly as oxides), CaO 8.89%, Zn 4.16%, MgO 3.64%, C 0.69%, SiO2 1.56%, Mn 0.57 %, K2O 0.281%, Na2O 0.251%, Al2O3 0.18%
- Lot/batch No.: 21.10.2009
- Expiration date of the lot/batch: unlimited
- Storage condition of test material: stored in PE container at room temperature

Method

Target gene:

gene for synthesis histidine or tryptophan

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine (Salmonella) and tryptophan (Escherichia) dependent

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:

With regard to poor solubility the test substance was dosed (50, 150, 500, 1500 and 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

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule which is compatible with the application of statistical methods (Dunkel V. C., Chu K.C.: 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; Claxton L. D. et al.: Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res., 189, 83 - 91, 1987). 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.

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:

not determined

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

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	134±10	-	30	160± 3	-
H2O	0	126± 4	-	30	143± 7	-
50	0	124±10	1.0	30	143±12	1.0
150	0	140±10	1.1	30	129± 8	0.9
500	0	142± 4	1.1	30	135± 2	0.9
1500	0	112± 5	0.9	30	152±19	1.1
5000	0	135±11	1.1	30	141± 6	1.0
AS/2-AF	0	475±21	3.8	20	1128±71	7.9
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	144± 8	-	30	164± 5	-
H2O	0	137± 4	-	30	157±13	-
50	0	131±15	1.0	30	169±11	1.1
150	0	145± 2	1.1	30	171±12	1.1
500	0	149± 3	1.1	30	173± 8	1.1
1500	0	150±18	1.1	30	161± 7	1.0
5000	0	148± 8	1.1	30	164±18	1.0
AS/2-AF	0	507± 9	3.7	20	778± 7	5.0
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	26± 1	-	30	18± 2	-
H2O	0	25± 2	-	30	16± 2	-
50	0	25± 5	1.0	30	15± 3	1.0
150	0	24± 2	1.0	30	20± 3	1.3
500	0	28± 6	1.1	30	17± 1	1.1
1500	0	20± 2	0.8	30	13± 2	0.8
5000	0	26± 2	1.0	30	9± 2	0.6
AS/2-AA	0	534±35	21.6	20	228± 5	14.5
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	23± 3	-	30	22± 1	-
H2O	0	24± 3	-	30	21± 2	-
50	0	30± 1	1.3	30	22± 3	1.0
150	0	26± 3	1.1	30	23± 1	1.1
500	0	27± 3	1.1	30	21± 6	1.0
1500	0	22± 1	0.9	30	20± 3	1.0
5000	0	25± 5	1.1	30	23± 2	1.1
AS/2-AA	0	423±26	17.9	20	223±10	10.5
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	26± 1	-	30	30± 4	-
H2O	0	27± 3	-	30	30± 7	-
50	0	33± 1	1.2	30	27± 1	0.9
150	0	29± 5	1.1	30	27± 3	0.9
500	0	26± 4	1.0	30	25± 4	0.8
1500	0	27± 3	1.0	30	30± 6	1.0
5000	0	23± 2	0.9	30	18± 2	0.6
NPD/2-AF	0	1382±22	51.8	20	1576±45	52.5
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	25± 0	-	30	40± 3	-
H2O	0	24± 1	-	30	33± 4	-
50	0	27± 6	1.1	30	31± 6	0.9
150	0	26± 4	1.1	30	34± 3	1.0
500	0	26± 0	1.1	30	29± 2	0.9
1500	0	21± 3	0.9	30	34± 3	1.0
5000	0	24± 4	1.0	30	30± 2	0.9
NPD/2-AF	0	1144±129	47.7	20	1351±22	40.5
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	13± 3	-	30	11± 2	-
H2O	0	13± 3	-	30	12± 2	-
50	0	15± 1	1.2	30	14± 2	1.2
150	0	11± 2	0.9	30	12± 0	0.9
500	0	12± 2	0.9	30	9± 1	0.7
1500	0	13± 1	1.1	30	11± 2	0.9
5000	0	12± 2	0.9	30	10± 2	0.8
9-AAc/2-AA	0	861±77	67.9	20	162± 7	13.1
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	11± 2	-	30	15± 4	-
H2O	0	10± 1	-	30	16± 1	-
50	0	11± 2	1.1	30	16± 1	1.0
150	0	9± 2	0.9	30	18± 4	1.1
500	0	9± 1	0.9	30	14± 3	0.9
1500	0	12± 1	1.3	30	13± 1	0.8
5000	0	9± 1	0.9	30	15± 1	1.0
9-AAc/2-AA	0	989±151	102.3	20	224±23	14.0
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	39± 5	-	100	32± 3	-
H2O	0	32± 5	-	100	31± 1	-
50	0	33± 2	1.0	100	38± 4	1.2
150	0	33± 4	1.0	100	43± 4	1.4
500	0	34± 8	1.1	100	37± 1	1.2
1500	0	35± 1	1.1	100	42± 3	1.3
5000	0	39± 2	1.2	100	37± 3	1.2
MNNG/2-AA	0	449± 1	14.0	100	162±12	5.2
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	37± 2	-	100	44± 2	-
H2O	0	43± 7	-	100	42± 1	-
50	0	37± 3	0.9	100	38± 3	0.9
150	0	39± 4	0.9	100	44± 4	1.0
500	0	42± 2	1.0	100	40± 1	1.0
1500	0	44± 4	1.0	100	47± 4	1.1
5000	0	40± 7	0.9	100	47± 4	1.1
MNNG/2-AA	0	474± 4	11.1	100	325±19	7.7

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the above-described experimental design, the test substance Dust, steelmaking 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 Dust, steelmaking 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 µg 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.

The test substance Dust, steelmaking was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.