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EC number: 940-591-0 | CAS number: -
Genetic toxicity: in vitro
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:
- 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed according to GLP
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
- 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:
- minor deviations not critical for the reliability of the study e.g. shaking instead of using a spatula
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Slags, ferrous metal, blast furnace
- EC Number:
- 266-002-0
- EC Name:
- Slags, ferrous metal, blast furnace
- Cas Number:
- 65996-69-2
- IUPAC Name:
- 266-002-0
- Details on test material:
- Name Slag, ferrous metal, blast furnace (air cooled - ABS), granular (8-11 mm)
Batch no. P2009 01862
Appearance solid
Composition see analytical certificate of FEhS-Institute
CAS No. 65996-69-2
EINECS-No. 266-002-0
Molecular formula not applicable (UVCB)
Molecular weight not applicable (UVCB)
Purity 100 w/w % slag
Homogeneity not stated
Vapour pressure extremely low (melting point >300°C)
Stability solid slag is stable at room temperature
Solubility slightly soluble in water
Production date not stated
Expiry date 12/2024
Storage Room temperature 20 ± 5°C
Constituent 1
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium, other: TA97a
- Details on mammalian cell type (if applicable):
- Salmonella typhimurium (all strains used) were obtained from Trinova Biochem and are stored as lyophilisate at 2 – 5 oC.
- Additional strain / cell type characteristics:
- other: mutations hisD6610, uvrB, pKM 101, rfa
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- other: mutations hisD3052, uvrB, pKM 101, rfa
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- other: mutations hisG46, uvrB, pKM 101, rfa
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- other: mutations hisG428, pKM 101, rfa
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- other: mutations hisG46, uvrB, rfa.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver enzyme mixture used for the test with metabolic activation
- Test concentrations with justification for top dose:
- One day before the start of each experiment, one pellet per strain was put into a culture vessel containing nutrient broth. For the incubation of strains TA97a, TA98, TA100, TA102; ampicilline was added to the nutrient broth (25 mg/L), for the incubation of strain TA102, tetracycline was added (20 mg/L) in addition to ampicilline. TA1535 was incubated without the addition of antibiotica
- Vehicle / solvent:
- no
Controls
- Untreated negative controls:
- yes
- Remarks:
- water
- Negative solvent / vehicle controls:
- no
- Positive controls:
- yes
- Remarks:
- Nitrophenylendiamine, 20 µg/plate: strains TA97a, TA98 and TA102 without S9. Sodium azide, 1 µg/plate: strains TA100 and TA1535 without S9. Benzo-a-pyrene, 20 µg/plate: strain TA98 with S9. 2-Amino-anthracene, 1 µg/plate: strains TA97a, TA100, TA102, TA15
- Positive control substance:
- other: Nitrophenylendiamine, 20 µg/plate: strains TA97a, TA98 and TA102 without S9. Sodium azide, 1 µg/plate: strains TA100 and TA1535 without S9. Benzo-a-pyrene, 20 µg/plate: strain TA98 with S9. 2-Amino-anthracene, 1 µg/plate: strains TA97a, TA100, TA102, TA15
- Details on test system and experimental conditions:
- other substances used
• Nutrient broth for overnight culture
• Isotonic sodium chloride solution for titre control
• Minimal Glucose Agar
• Biotin-Agar
• Histidin-Biotin-Agar
• Ampicilline-Agar
• Ampicilline-Tetracycline-Agar
• Nutrient-Agar
• Histidin-Biotin-solution 0.5 millimolar for Top-Agar
• Histidin-Biotin-solution 0.5 millimolar/5 millimolar for Maximal-Softagar
• Basis for Top-Agar and Maximal-Soft-Agar
• Salt solution for S9-Mix
• Phosphate buffer for S9-Mix
• NADP-solution for S9-Mix
• Glucose-6-phosphate-solution for S9-Mix
Genotype Confirmation
The following genotype specifications of the test system were routinously tested every three months:
• histidine deficiency
• ampicilline resistance (pKM 101, for TA97a, TA98, TA100, TA102; control : TA 1535)
• Sensitivity against crystal violet (deep rough, for TA 97a, TA98, TA100, TA102, TA1535)
• UV-sensitivity (uvrB, only TA102 may show growth)
• ampicilline-tetracycline-resistance (pAQ1, TA102) - Evaluation criteria:
- A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
- Statistics:
- The mean values and standard deviations of each fourfold determination are calculated as well as the increase factor of revertant induction (revertants divided by spontaneous revertants) of the test item solutions and the positive controls.
Results and discussion
Test results
- Species / strain:
- S. typhimurium, other: TA97a, TA98 TA100, TA102, TA1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not applicable
- 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
All relevant data are documented in the test report
Determination of Titre
The determination of titre should give a number of at least 109cells/mL, correlating to 100 colonies/plate
Titre Values (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
Repl. 1 (-S9) |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
Repl. 2 (-S9) |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
Mean |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
sd |
0 |
0 |
0 |
0 |
0 |
Repl. 3 (+S9) |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
Repl. 4 (+S9) |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
Mean |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
sd |
0 |
0 |
0 |
0 |
0 |
Assessment |
ok |
ok |
ok |
ok |
ok |
Toxicity Control
The test item is considered non-toxic, if the quotient titre/tox is below 2.
Table 16.3‑a 10.000µg/plate on maximal-soft-agar with culture diluted by 106
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
Repl. 1 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
Repl. 2 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
Mean |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
sd |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
Repl. 3 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
Repl. 4 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
Mean |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
> 1000 |
sd |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
Titre/Tox |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
Historic Data
Comparison with historical data (conclusion: results are valid)
Strain |
|
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
|
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
H2O |
Mean |
107 |
112 |
9 |
9 |
123 |
120 |
255 |
258 |
13 |
14 |
Min |
100 |
97 |
6 |
5 |
104 |
97 |
128 |
157 |
6 |
7 |
|
Max |
114 |
127 |
17 |
15 |
161 |
175 |
306 |
345 |
23 |
19 |
|
Exp 1 |
108 |
110 |
14 |
11 |
101 |
109 |
156 |
193 |
18 |
12 |
|
Exp 2 |
121 |
121 |
13 |
10 |
130 |
132 |
193 |
212 |
4 |
7 |
|
DMSO |
Mean |
107 |
108 |
10 |
9 |
108 |
110 |
254 |
234 |
13 |
13 |
Min |
96 |
90 |
6 |
6 |
93 |
100 |
95 |
111 |
6 |
7 |
|
Max |
114 |
116 |
15 |
17 |
134 |
128 |
313 |
305 |
21 |
19 |
|
Exp 1 |
114 |
118 |
19 |
14 |
106 |
109 |
236 |
195 |
16 |
12 |
|
Exp 2 |
115 |
118 |
15 |
12 |
119 |
118 |
200 |
182 |
6 |
7 |
|
Pos. Contr. |
Mean |
541 |
513 |
351 |
122 |
665 |
704 |
1112 |
1106 |
225 |
121 |
Min |
339 |
271 |
115 |
50 |
386 |
499 |
913 |
785 |
116 |
105 |
|
Max |
903 |
830 |
978 |
259 |
1212 |
1273 |
1285 |
1345 |
374 |
143 |
|
Exp 1 |
326 |
276 |
156 |
154 |
667 |
698 |
1059 |
1277 |
152 |
153 |
|
Exp 2 |
314 |
284 |
160 |
150 |
531 |
572 |
1129 |
1189 |
156 |
143 |
|
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
Slag, ferrous metal, blast furnace (air cooled - ABS), granular (8-11 mm) is not mutagenic under the conditions of the test. - Executive summary:
For determination of the mutagenic potential of slags, ferrous metal, blast furnace (air cooled - ABS), granular (8-11 mm) the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 was performed. As the slags are inorganic solids, a leachate of the slags was prepared as prescribed by DIN 38414-S4 (100 g/L).
Two experiments were performed:
In the first experiment, 5 concentrations of the leachate (ranging from 10060 to 101 µg/plate, nominal concentration) were used. 5 genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the slags both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method.
In the second experiment, 5 concentrations of the leachate (ranging from 10160 to 635 µg/plate) were tested using the pre-incubation method.
Both experiments yielded the same results: None of the concentrations caused any significant increase in the number of revertant colonies in the tested bacterial strains. The slags had no mutagenic effect towards Salmonella typhimurium, strainsTA 97a, TA 98, TA 100, TA 102 and TA 1535. The slags exhibited no sign of toxicity towards the bacteria. No inconsistencies were observed in the sterility control and in the determination of the titre. The numbers of spontaneous revertants of the negative controls were in the normal range, and all positive controls showed mutagenic effects with and without metabolic activation.
Slags, ferrous metal, blast furnace (air cooled - ABS), granular (8-11 mm) are not mutagenic under the conditions of the Ames test.
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