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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:
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

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)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
AS
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
AAc

Migrated to IUCLID6: hydrochloride monohydrate
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
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene (CASRN: 153-78-6)
Remarks:
AF
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (CASRN: 613-13-8)
Remarks:
AA
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
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:
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.