Registration Dossier
Registration Dossier
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EC number: 208-865-8 | CAS number: 544-19-4
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:
- 2019-05-22 to 2019-06-27
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- 2000
- Qualifier:
- according to guideline
- Guideline:
- other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)
- Version / remarks:
- 2012
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Copper diformate
- EC Number:
- 208-865-8
- EC Name:
- Copper diformate
- Cas Number:
- 544-19-4
- Molecular formula:
- CH2O2.1/2Cu
- IUPAC Name:
- copper diformate
- Test material form:
- solid: particulate/powder
- Details on test material:
- blue in color
Constituent 1
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1537
- Remarks:
- his C 3076; rfa-; uvrB-: frame shift
- Species / strain / cell type:
- S. typhimurium TA 98
- Remarks:
- his D 3052; rfa-; uvrB-;R-factor; frame shift
- Species / strain / cell type:
- S. typhimurium TA 1535
- Remarks:
- his G 46; rfa-; uvrB-: base-pair substitution
- Species / strain / cell type:
- S. typhimurium TA 100
- Remarks:
- his G 46; rfa-; uvrB-;R-factor; base-pair substitution
- Species / strain / cell type:
- E. coli WP2 uvr A
- Remarks:
- trp-; uvrA-: base-pair substitution
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (Experiment I: plate incorporation method)
15, 50, 150, 500, 1500 and 5000 µg/plate (Experiment II: pre-incubation method)
0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate (Experiment II (repeat): pre-incubation method, presence of S9) - Vehicle / solvent:
- sterile distilled water
Negative (untreated) controls were also performed on the same day as the mutation test.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 3 (Experiment I, Experiment II, Experiment II r(epeat))
- The sterility controls: yes
The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix; Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and The maximum dosing solution of the test item in the absence of S9-mix only (tested in singular prior to Experiment 1).
METHOD OF TREATMENT/ EXPOSURE:
Experiment I plate incorporation
Experiment II preincubation
TREATMENT Experiment II:
- Preincubation period, if applicable: at 37 ± 3 °C for 20 minutes
- Exposure duration/duration of treatment: at 37 ± 3 °C for between 48 and 72 hours
METHODS FOR MEASUREMENT OF CYTOTOXICITY
bacterial background lawns and/or a reduction in revertant counts
- Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that are statistically significant but are within the in-house historical vehicle/untreated control range are not reported in the tables section.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment II with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment II with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment II with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment II with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment II with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
STUDY RESULTS
- Concurrent vehicle negative and positive control data
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
Experiment I
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
A test item colouration (light blue in appearance for all strains except TA1537 which showed a light brown colouration) was noted at and above 1500 µg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). One statistically significant value was noted (TA100 at 5000 µg/plate in the absence of metabolic activation (S9-mix)), however, this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.
Experiment II repeat
There was no visible reduction in the growth of the bacterial background lawn at any dose level, in the absence of metabolic activation (S9-mix). However, toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns and/or a reduction in revertant counts, was observed with test item exposure to all tester strains dosed in the presence of metabolic activation from 500 µg/plate.
A test item colouration (light blue in appearance for all strains except TA1537 which showed a light brown colouration) was noted at and above 1500 µg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
No biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). Three statistically significant values were noted (TA100 at 150, 500 and 5000 µg/plate in the absence of metabolic activation (S9-mix)), however, these responses were within the in-house historical vehicle/untreated control range for the strain and were, therefore considered of no biological relevance.
HISTORICAL CONTROL DATA
Yes
Any other information on results incl. tables
Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From: 11 June 2019 |
To: 14 June 2019 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
151 116 140 |
(136) 17.9# |
10 8 13 |
(10) 2.5 |
25 22 30 |
(26) 4.0 |
17 17 29 |
(21) 6.9 |
8 10 7 |
(8) 1.5 |
||
1.5 µg |
132 111 131 |
(125) 11.8 |
9 19 6 |
(11) 6.8 |
19 27 26 |
(24) 4.4 |
20 24 23 |
(22) 2.1 |
14 12 9 |
(12) 2.5 |
||
5 µg |
150 151 146 |
(149) 2.6 |
7 14 12 |
(11) 3.6 |
23 15 27 |
(22) 6.1 |
16 32 21 |
(23) 8.2 |
18 9 13 |
(13) 4.5 |
||
15 µg |
180 134 138 |
(151) 25.5 |
12 19 17 |
(16) 3.6 |
27 18 30 |
(25) 6.2 |
22 23 15 |
(20) 4.4 |
11 16 14 |
(14) 2.5 |
||
50 µg |
135 156 162 |
(151) 14.2 |
13 19 12 |
(15) 3.8 |
18 24 16 |
(19) 4.2 |
23 15 19 |
(19) 4.0 |
15 14 13 |
(14) 1.0 |
||
150 µg |
158 153 171 |
(161) 9.3 |
13 12 15 |
(13) 1.5 |
22 15 40 |
(26) 12.9 |
24 24 36 |
(28) 6.9 |
16 23 6 |
(15) 8.5 |
||
500 µg |
163 144 162 |
(156) 10.7 |
12 13 12 |
(12) 0.6 |
21 20 27 |
(23) 3.8 |
19 31 21 |
(24) 6.4 |
8 8 16 |
(11) 4.6 |
||
1500 µg |
161 164 154 |
(160) 5.1 |
11 8 14 |
(11) 3.0 |
19 32 24 |
(25) 6.6 |
16 25 19 |
(20) 4.6 |
15 14 15 |
(15) 0.6 |
||
5000 µg |
167 170 170 |
(169) 1.7 |
10 9 10 |
(10) 0.6 |
36 18 33 |
(29) 9.6 |
14 17 14 |
(15) 1.7 |
15 17 6 |
(13) 5.9 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
536 551 485 |
(524) 34.6 |
332 538 332 |
(401) 118.9 |
633 596 561 |
(597) 36.0 |
123 118 145 |
(129) 14.4 |
139 114 147 |
(133) 17.2 |
|||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
# Standard deviation
Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)
Test Period |
From: 11 June 2019 |
To: 14 June 2019 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
145 173 160 |
(159) 14.0# |
15 13 13 |
(14) 1.2 |
44 34 35 |
(38) 5.5 |
20 34 20 |
(25) 8.1 |
12 7 16 |
(12) 4.5 |
||
1.5 µg |
152 136 167 |
(152) 15.5 |
7 8 14 |
(10) 3.8 |
28 29 29 |
(29) 0.6 |
24 30 38 |
(31) 7.0 |
17 12 13 |
(14) 2.6 |
||
5 µg |
149 145 159 |
(151) 7.2 |
19 11 17 |
(16) 4.2 |
23 29 40 |
(31) 8.6 |
21 38 22 |
(27) 9.5 |
15 20 14 |
(16) 3.2 |
||
15 µg |
169 148 185 |
(167) 18.6 |
7 12 14 |
(11) 3.6 |
26 20 26 |
(24) 3.5 |
27 23 19 |
(23) 4.0 |
13 10 16 |
(13) 3.0 |
||
50 µg |
147 158 152 |
(152) 5.5 |
15 15 9 |
(13) 3.5 |
C 31 31 |
(31) 0.0 |
21 22 25 |
(23) 2.1 |
14 12 13 |
(13) 1.0 |
||
150 µg |
133 130 160 |
(141) 16.5 |
20 7 11 |
(13) 6.7 |
18 29 28 |
(25) 6.1 |
24 20 22 |
(22) 2.0 |
13 13 11 |
(12) 1.2 |
||
500 µg |
159 159 150 |
(156) 5.2 |
11 8 13 |
(11) 2.5 |
25 27 38 |
(30) 7.0 |
24 14 24 |
(21) 5.8 |
11 18 16 |
(15) 3.6 |
||
1500 µg |
141 148 176 |
(155) 18.5 |
15 11 11 |
(12) 2.3 |
45 41 42 |
(43) 2.1 |
18 35 32 |
(28) 9.1 |
11 14 11 |
(12) 1.7 |
||
5000 µg |
155 155 158 |
(156) 1.7 |
14 15 10 |
(13) 2.6 |
34 16 45 |
(32) 14.6 |
19 24 25 |
(23) 3.2 |
13 17 14 |
(15) 2.1 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1666 1806 1749 |
(1740) 70.4 |
304 322 309 |
(312) 9.3 |
233 204 240 |
(226) 19.1 |
191 188 196 |
(192) 4.0 |
224 217 224 |
(222) 4.0 |
|||
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
C Contaminated
# Standard deviation
Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)
Test Period |
From: 17 June 2019 |
To: 20 June 2019 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
127 134 129 |
(130) 3.6# |
13 16 16 |
(15) 1.7 |
28 21 34 |
(28) 6.5 |
21 20 22 |
(21) 1.0 |
13 17 11 |
(14) 3.1 |
||
15 µg |
158 130 151 |
(146) 14.6 |
16 14 11 |
(14) 2.5 |
40 14 31 |
(28) 13.2 |
18 16 20 |
(18) 2.0 |
15 14 9 |
(13) 3.2 |
||
50 µg |
136 152 170 |
(153) 17.0 |
21 23 18 |
(21) 2.5 |
26 35 34 |
(32) 4.9 |
21 25 20 |
(22) 2.6 |
13 17 10 |
(13) 3.5 |
||
150 µg |
154 156 172 |
(161) 9.9 |
21 15 19 |
(18) 3.1 |
36 34 27 |
(32) 4.7 |
22 27 23 |
(24) 2.6 |
14 16 14 |
(15) 1.2 |
||
500 µg |
175 156 149 |
(160) 13.5 |
18 28 29 |
(25) 6.1 |
31 19 24 |
(25) 6.0 |
13 22 23 |
(19) 5.5 |
13 16 12 |
(14) 2.1 |
||
1500 µg |
175 136 138 |
(150) 22.0 |
27 19 9 |
(18) 9.0 |
46 50 18 |
(38) 17.4 |
25 23 21 |
(23) 2.0 |
13 14 10 |
(12) 2.1 |
||
5000 µg |
168 169 149 |
(162) 11.3 |
18 29 23 |
(23) 5.5 |
38 19 17 |
(25) 11.6 |
27 20 C |
(24) 4.9 |
8 4 8 |
(7) 2.3 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
638 610 568 |
(605) 35.2 |
687 747 732 |
(722) 31.2 |
720 771 689 |
(727) 41.4 |
197 217 255 |
(223) 29.5 |
279 218 145 |
(214) 67.1 |
|||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
C Contaminated
# Standard deviation
Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)
Test Period |
From: 24 June 2019 |
To: 27 June 2019 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
127 91 115 |
(111) 18.3# |
11 10 14 |
(12) 2.1 |
32 31 32 |
(32) 0.6 |
27 32 20 |
(26) 6.0 |
13 13 19 |
(15) 3.5 |
||
0.5 µg |
95 113 104 |
(104) 9.0 |
10 6 10 |
(9) 2.3 |
44 19 31 |
(31) 12.5 |
25 23 23 |
(24) 1.2 |
11 11 11 |
(11) 0.0 |
||
1.5 µg |
99 113 106 |
(106) 7.0 |
19 6 12 |
(12) 6.5 |
32 30 29 |
(30) 1.5 |
35 23 26 |
(28) 6.2 |
15 16 10 |
(14) 3.2 |
||
5 µg |
118 115 101 |
(111) 9.1 |
9 13 12 |
(11) 2.1 |
33 28 26 |
(29) 3.6 |
31 24 25 |
(27) 3.8 |
10 12 14 |
(12) 2.0 |
||
15 µg |
91 111 97 |
(100) 10.3 |
15 16 6 |
(12) 5.5 |
32 35 23 |
(30) 6.2 |
18 20 20 |
(19) 1.2 |
7 8 14 |
(10) 3.8 |
||
50 µg |
97 112 116 |
(108) 10.0 |
10 4 13 |
(9) 4.6 |
19 21 30 |
(23) 5.9 |
21 25 30 |
(25) 4.5 |
7 7 13 |
(9) 3.5 |
||
150 µg |
109 118 120 |
(116) 5.9 |
12 7 7 |
(9) 2.9 |
28 25 26 |
(26) 1.5 |
27 26 17 |
(23) 5.5 |
10 7 13 |
(10) 3.0 |
||
500 µg |
103 S 123 S 95 S |
(107) 14.4 |
12 S 12 S 9 S |
(11) 1.7 |
29 S 17 S 22 S |
(23) 6.0 |
22 S 26 S 35 S |
(28) 6.7 |
8 S 17 S 8 S |
(11) 5.2 |
||
1500 µg |
103 S 89 S 95 S |
(96) 7.0 |
10 S 11 S 9 S |
(10) 1.0 |
20 S 23 S 23 S |
(22) 1.7 |
20 S 24 S 21 S |
(22) 2.1 |
10 S 10 S 6 S |
(9) 2.3 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1179 1181 1116 |
(1159) 37.0 |
224 281 290 |
(265) 35.8 |
105 106 100 |
(104) 3.2 |
154 191 196 |
(180) 22.9 |
206 207 180 |
(198) 15.3 |
|||
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
S Sparse bacterial background lawn
# Standard deviation
Applicant's summary and conclusion
- Conclusions:
- Copper Diformate was considered to be non-mutagenic under the conditions of this test.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
