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EC number: 300-491-4 | CAS number: 93940-93-3
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-10-27 to 2016-11-21
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- May 30, 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- EPA 712-C-98-247, August 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper
- EC Number:
- 300-491-4
- EC Name:
- Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper
- Cas Number:
- 93940-93-3
- Molecular formula:
- C10H20CuN2O6
- IUPAC Name:
- bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper
- Test material form:
- solid: particulate/powder
Constituent 1
Method
- Target gene:
- histidine
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix
- Test concentrations with justification for top dose:
- Pre-experiment (plate incorporation): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment I (plate incorporation test with and without rat liver S9): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II (pre-incubation test with and without rat liver S9):3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Aqua dest.
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- Aqua dest.; negative control =solvent control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Aqua dest.; negative control =solvent control
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 100, TA 1535 without metabolic activation dissolved in Aqua dest.
- Positive control substance:
- sodium azide
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 98, TA 1537 without metabolic activation dissolved in DMSO
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine (4-NOPD)
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 102 without metabolic activation dissolved in Aqua dest.
- Positive control substance:
- methylmethanesulfonate
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102 with metabolic activation dissolved in DMSO
- Positive control substance:
- other: 2-aminoanthracene (2-AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: pre-incubation
DURATION
Experiment I:
- Preincubation period: No
- Exposure duration: at least 48 h in the dark at 37 °C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable
Experiment II:
- Preincubation period: 60 min at 37°C
- Exposure duration: 60 min at 37°C and at least 48 h in the dark at 37°C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable
SELECTION AGENT (mutation assays): histidine (overlay agar)
NUMBER OF REPLICATIONS: 3 (in two cases only two plates were evaluated)
DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control - Evaluation criteria:
- The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. - Statistics:
- According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Experiment I
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Experiment I
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Experiment I
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Experiment I
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Remarks:
- Experiment I
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Experiment II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Experiment II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of 1000 µg/plate and higher (with metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Experiment II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of 316 µg/plate and higher (with metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Experiment II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of 316 µg/plate and higher (with metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Remarks:
- Experiment II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at a concentration of 5000 µg/plate (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
RANGE-FINDING/SCREENING STUDIES:
see any other information on results
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) historical control data: The negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2013 -2015)):
-S9 + S9 (rat liver)
min max min max
TA 98 13 54 13 61
TA 100 49 139 67 162
TA 1535 4 39 4 32
TA 1537 2 35 3 36
TA 102 141 472 91 586
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
- Other observations when applicable: Toxic effects of the test item were noted in all tester strains used in experiment II at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation), depending on the particular tester strain.
Any other information on results incl. tables
Pre-experiment:
Substance |
Dose (μg/plate) |
TA 98 Mutation Factor [toxicity]* |
TA 100 Mutation Factor [toxicity]* |
||
without S9 |
with S9 |
without S9 |
with S9 |
||
Solvent Control (A. dest) |
|
1.0 |
1.0 |
1.0 |
1.0 |
4-NOPD |
10.0 |
25.8 |
- |
- |
- |
NaN3 |
10.0 |
- |
- |
6.1 |
- |
2-AA |
2.50 |
- |
53.2 |
- |
11.6 |
Test Item
|
3.16 |
1.1 |
0.9 |
0.9 |
0.9 |
10.0 |
1.1 |
0.8 |
0.9 |
0.8 |
|
31.6 |
0.9 |
0.7 |
1.0 |
1.1 |
|
100 |
1.1 |
0.7 |
1.2 |
1.1 |
|
316 |
1.2 |
0.8 |
1.1 |
1.1 |
|
1000 |
1.3 |
0.9 |
1.2 |
1.1 |
|
2500 |
1.0 |
1.1 |
1.2 |
1.3 |
|
5000 |
1.3 |
0.8 |
1.3 |
1.2 |
* [toxicity parameter]: B = Background lawn reduced; N = No background lawn
Experiment I
TA 98 | TA 100 | TA 1535 | TA 1537 | TA 102 | |||||||||||||||||
Revertant colonies per plate | Mutation factor | Revertant colonies per plate | Mutation factor | Revertant colonies per plate | Mutation factor | Revertant colonies per plate | Mutation factor | Revertant colonies per plate | Mutation factor | ||||||||||||
Treatment | Dose (µg)/plate | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
A. Dest | 22 | 31 | 1 | 1 | 128 | 128 | 1 | 1 | 20 | 17 | 1 | 1 | 7 | 9 | 1 | 1 | 297 | 336 | 1 | 1 | |
Test item | 31.6 | 20 | 22 | 0.9 | 0.7 | 128 | 135 | 1 | 1.1 | 19 | 26 | 0.9 | 1.6 | 9 | 8 | 1.2 | 0.9 | 244 | 363 | 0.8 | 1.1 |
Test item | 100 | 25 | 21 | 1.1 | 0.7 | 148 | 143 | 1.2 | 1.1 | 46 | 28 | 2.2 | 1.7 | 10 | 6 | 1.4 | 0.6 | 260 | 403 | 0.9 | 1.2 |
Test item | 316 | 326 | 26 | 1.2 | 0.8 | 137 | 135 | 1.1 | 1.1 | 24 | 36 | 1.2 | 2.1 | 8 | 6 | 1 | 0.6 | 307 | 372 | 1 | 1.1 |
Test item | 1000 | 28 | 28 | 1.3 | 0.9 | 158 | 141 | 1.2 | 1.1 | 46 | 28 | 2.2 | 1.7 | 11 | 10 | 1.5 | 1.1 | 278 | 327 | 0.9 | 1 |
Test item | 2500 | 22 | 33 | 1 | 1.1 | 153 | 165 | 1.2 | 1.3 | 25 | 26 | 1.2 | 1.5 | 9 | 9 | 1.3 | 1 | 288 | 372 | 1 | 1.1 |
Test item | 5000 | 30 | 24 | 1.3 | 0.8 | 164 | 148 | 1.3 | 1.2 | 24 | 26 | 1.2 | 1.6 | 8 | 7 | 1 | 0.7 | 317 | 404 | 1.1 | 1.2 |
4-NOPD/NaN3/MMS | 10 | 576 | - | 25.8 | - | 776 | - | 6.1 | - | 1009 | - | 49.6 | - | 91 | - | 12.4 | - | 1303 | - | 4.4 | - |
2-AA | 2.5 | - | 1632 | - | 53.2 | - | 1488 | - | 11.6 | - | 211 | - | 12.7 | - | 223 | - | 24.7 | - | 757 | - | 2.3 |
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I.
Experiment II:
TA 98 | TA 100 | TA 1535 | TA 1537 | TA 102 | |||||||||||||||||
Revertant colonies per plate | Mutation factor | Revertant colonies per plate | Mutation factor | Revertant colonies per plate | Mutation factor | Revertant colonies per plate | Mutation factor | Revertant colonies per plate | Mutation factor | ||||||||||||
Treatment | Dose (µg)/plate | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
A. Dest | 25 | 29 | 1 | 1 | 133 | 95 | 1 | 1 | 18 | 19 | 1 | 1 | 11 | 10 | 1 | 1 | 267 | 255 | 1 | 1 | |
Test item | 31.6 | 26 | 21 | 1.1 | 0.7 | 126 | 122 | 1 | 1.3 | 25 | 29 | 1.4 | 1.5 | 12 | 8 | 1.1 | 0.8 | 298 | 377 | 1.1 | 1.5 |
Test item | 100 | 22 | 35 | 0.9 | 1.2 | 149 | 124 | 1.1 | 1.3 | 27 | 20 | 1.5 | 1 | 12 | 8 | 1.1 | 0.7 | 253 | 338 | 0.9 | 1.3 |
Test item | 316 | 26 | 19 | 1 | 0.7 | 149 | 112 | 1.1 | 1.2 | 20 | 22 | 1.1 | 1.2 | 10 | 5 | 0.9 | 0.5 | 267 | 190 | 1 | 0.7 |
Test item | 1000 | 23 | 29 | 0.9 | 1 | 90 | 106 | 0.7 | 1.1 | 31 | 18 | 1.7 | 0.9 | 7 | 7 | 0.6 | 0.7 | 266 | 258 | 1 | 1 |
Test item | 2500 | 18 | 14 | 0.7 | 0.5 | 123 | 103 | 0.9 | 1.1 | 22 | 7 | 1.2 | 0.4 | 9 | 7 | 0.8 | 0.7 | 277 | 183 | 1 | 0.7 |
Test item | 5000 | 25 | 3 | 1 | 0.1 | 79 | 39 | 0.6 | 0.4 | 19 | 5 | 1.1 | 0.3 | 7 | 3 | 0.6 | 0.3 | 155 | 149 | 0.6 | 0.6 |
4-NOPD/NaN3/MMS | 10 | 545 | - | 22.1 | - | 890 | - | 6.7 | - | 947 | - | 52.6 | - | 128 | - | 11.7 | - | 1257 | - | 4.7 | - |
2-AA | 2.5 | - | 125 | - | 4.3 | - | 1032 | - | 10.9 | - | 82 | - | 4.2 | - | 103 | - | 9.9 | - | 710 | - | 2.8 |
Due to more sensitive pre-incubation method, in experiment II toxic effects of the test item were seen in most tester strains.
- In tester strain TA 98 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (with metabolic activation).
- In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation).
- In tester strains TA 1535 and TA 1537 toxic effects of the test item were seen at concentrations of 316 µg/plate and higher (with metabolic activation).
- In tester strain TA 102 toxic effects of the test item were observed at a concentration of 5000 µg/plate (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).
Applicant's summary and conclusion
- Conclusions:
- During the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
- Executive summary:
In order to investigate the ability of the test substance to induce gene mutations, a bacterial reverse mutation assay (Ames) according to OECD 471 was performed. Two experiments were ran: the plate incorporation test (experiment I) and the pre-incubation test (experiment II) with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation (S9 ). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in both experiments: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I.
Due to more sensitive pre-incubation method, in experiment II toxic effects of the test item were seen in most tester strains. In tester strain TA 98 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were seen at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA 102 toxic effects of the test item were observed at a concentration of 5000 µg/plate (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
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