Registration Dossier
Registration Dossier
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Copper monoglycinate sulfate is not expected to be mutagenic in the bacterial reverse mutation assay.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-12-03 to 2020-01-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 21 July, 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his locus
- 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:
- Type and composition of metabolic activation system:
S9 liver Mix
- source of S9 : S9 was obtained by Trinova Biochem GmbH, Gießen.
- method of preparation of S9 mix: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.
- concentration or volume of S9 mix and S9 in the final culture medium: 500µL - Test concentrations with justification for top dose:
- nominal concentrations: 0, 50, 150, 500, 1500, 5000 µg/plate Experiment 1 and 0, 78, 156, 313, 625, 1250, 2500, 5000 /plate Experiment 2
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO; demineralized water
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, dimethyl sulfoxide (DMSO), acetone
The solid test item is not sufficiently soluble in any of the solvents. Based on the non-GLP pre-test, a test item suspension in demin. water was used, because this solvent shows the most consistent suspension with the test item and does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. The following nominal concentrations were prepared for the experiments 1b and 1d: 5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate. The following suspensions were stirred during the experiment: 5000 µg/plate, 1500 µg/plate, 500 µg/plate. The lower concentrations were dissolved completely and stirring was not necessary anymore. On the day of the start of the second experiment (2b), a test item stock solution containing 50 ± 5 g/L suspended in demin. water was prepared.
The following nominal concentrations were prepared for the second experiment: 5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 313 µg/plate, 156 µg/plate and 78 µg/plate. The suspensions were stirred during the experiment, except from 156 µg/plate and 78 µg/plate, they were dissolved completely and stirring was not necessary anymore.
DMSO was chosen due to the solubility of the positive controls 4-Nitro-1,2-phenylene diamine, benzo-a-pyrene and 2-amino-anthracene. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- other: 4-Nitro-1,2-phenylene diamine: without metabolic activation, TA98, TA102, TA1537, 20 µg (TA98) and 30 µg (TA102 and TA1537) 2-Amino-anthracene: with metabolic activation, TA 100, TA102, TA1535, TA1537, 1µg (TA100, TA1535) and 2.4 µg (TA102, TA1537)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate for each with and without metabolic acitvation
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): > E+09 cells/mL
- Test substance added in agar (plate incorporation)and in the second experiment with preincubation
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
- Any supplementary information relevant to cytotoxicity: No cytotoxicity nor precipitation occurred with the test item during the experimental time.
- Rationale for test conditions:
- as recommended by OECD Testguideline 471
- Evaluation criteria:
- The colonies were counted visually and the numbers were recorded. A substance is considered to be mutagenic, if a reproducible increase with or without metabolic activation of revertant colonies per plate exceeding an increase factor of 2 for the bacteria strains TA98, TA100, TA102, TA1535 and TA1537 compared to vehicle controls 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. A substance is not mutagenic if it does not meet these criteria. If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed.
- Statistics:
- The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other: The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other: The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Key result
- Species / strain:
- S. typhimurium TA 102
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other: The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other:
- Remarks:
- The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Key result
- Species / strain:
- S. typhimurium TA 100
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other: The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Water solubility: Not soluble in demineralized water, suspensions were used in the experiments.
RANGE-FINDING/SCREENING STUDIES (if applicable):
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, dimethyl sulfoxide (DMSO), acetone.
The solid test item is not sufficiently soluble in any of the solvents. Based on the non-GLP pre-test, a test item suspension in demin. water was used, because this solvent shows the most consistent suspension with the test item and does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. The following nominal concentrations were prepared for the experiments 1b and 1d: 5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate. The following suspensions were stirred during the experiment: 5000 µg/plate, 1500 µg/plate, 500 µg/plate. The lower concentrations were dissolved completely and stirring was not necessary anymore. On the day of the start of the second experiment (2b), a test item stock solution containing 50 ± 5 g/L suspended in demin. water was prepared.
The following nominal concentrations were prepared for the second experiment: 5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 313 µg/plate, 156 µg/plate and 78 µg/plate. The suspensions were stirred during the experiment, except from 156 µg/plate and 78 µg/plate, they were dissolved completely and stirring was not necessary anymore.no soluble in demineralized water
STUDY RESULTS
- Concurrent vehicle negative and positive control data Please refer to 'Any other infomation on results incl. tables'
Ames test:
- Signs of toxicity : No
- Individual plate counts : Please refer to 'Any other information on results incl. tables'.
- Mean number of revertant colonies per plate and standard deviation: Please refer to 'Any other information on results incl. tables'.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to 'Any other information on results incl. tables'.
- Negative (solvent/vehicle) historical control data: Please refer to 'Any other information on results incl. tables'. - Conclusions:
- There was no evidence of induced mutant colonies over background, when bis(glycinato)copper was tested with and without metabolic activation up to the recommended limit concentration (5000 µg/plate).
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD guideline 471 and EU Method B.14 (Version Commission Directive 92/69/EEC), strains TA1535, TA 1537, TA 102, TA 100 and TA 98 of S. typhimurium were exposed to bis(glycinato)copper. The test was performed with concentrations up to the recommended limit concentration of 5000 µg/plate in the absence and the presence of mammalian metabolic activation (S9 -mix).
No evidence of biologically significant mutagenic activity of the test item was found in the presence and absence of metabolic activation, up to the limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed.
There was no evidence of induced mutant colonies over background. Based on the results of this study, it is concluded that bis(glycinato)copper is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- not reported
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- The study was conducted in 1984.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Test material was supplied from the Japan Food Additives Association, Tokyo, at the request of the Ministry of Health and Welfare of Japan, where the purity and quality of each sample were checked.
- Target gene:
- histidine operon
- Species / strain / cell type:
- S. typhimurium TA 1537
- Species / strain / cell type:
- S. typhimurium TA 1535
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium, other: TA92
- Species / strain / cell type:
- S. typhimurium, other: TA94
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- method of preparation of S9 mix: The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co.) pre-treated 5 days before with polychlorinated biphenyls (500 mg/kg body weight of Kanechlor KC-400 in olive oil, ip). The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4mM-NADPH, 4mM-NADH, 33mM-KCl, 8 mM-MgCI2, 100 mM-phosphate buffer (pH 7.4) and 3.75 ml S-9 (129 mg protein) in a total volume of 12.5 ml. - Test concentrations with justification for top dose:
- 35000 µg/plate (crystal) and 33000 µg/plate (powder), 6 doses. The maximum dose represents the highest non-cytotoxic dose used in the experiment.
- Vehicle / solvent:
- phosphate buffer
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: Fourteen out of 200 tested substances in the Ames assay were identified as mutagenic, which proves the validity of the system.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION:
-Preincubation period: 20 minutes
-Exposure duration: 48 hours
NUMBER OF PLATES : 2 - Evaluation criteria:
- The result was considered positive, if the number of colonies found was twice the number in the control.
- Statistics:
- not reported
- Key result
- Species / strain:
- S. typhimurium, other: TA94
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium, other: TA92
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- The test item was not mutagenic in the bacterial reverse mutation assay.
- Executive summary:
In the study by Ishidate et al. (1984), the mutagenic potential of glycine was examined in a bacterial reverse mutation assay according to the method described by Ames et al. (1975).
6 tester strains of Salmonella typhimurium (TA92, TA1535, TA100, TA1537, TA94 and TA98) were treated with the test material, both with and without metabolic activation. 6 doses were applied, and the maximum doses were 35000 µg/plate (crystal) and 33000 µg/plate (powder), which represent the highest non-cytotoxic dose used in the experiment.
No significant increases in the frequency of revertant colonies were observed for any of the bacterial strains at any dose of glycine, either with or without metabolic activation. No information on the validity of vehicle and positive controls is reported.
In conclusion, glycine was not mutagenic in the bacterial reverse mutation assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- other: reported in the European Union Risk Assessment Report (2008) on copper and copper compounds.
- Adequacy of study:
- supporting study
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- GLP compliance:
- not specified
- Remarks:
- No information on GLP provided.
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- 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 S-9
- Test concentrations with justification for top dose:
- Doses up to 1000 µg/plate. The highest concentration caused cytotoxicity.
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- not specified
- Details on test system and experimental conditions:
- Duplicate experiments.
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- According to the EU RAR (2008), copper sulfate is negative in the bacterial reverse mutation assay.
- Executive summary:
The EU RAR (2008) reports unpublished data from an Ames test using copper sulfate pentahydrate which are considered as reliable without restriction. In the respective study, copper sulfate was tested in duplicate experiments in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in the presence and absence of metabolic activation (rat liver S-9), at doses up to 1000 µg/plate. The highest concentration caused cytotoxicity. In the second experiment, a one-hour pre-incubation step was employed. In all strains, copper sulfate failed to cause a dose-dependent, significant increase in revertant colonies over the control level, with or without metabolic activation. This study, which was conducted according to EU Annex V Test Guidelines (method B.14), clearly shows copper sulfate to be negative in the Ames test.
Two further, less reliable studies are discussed. A negative result was reported for copper sulfate in the Ames test using Salmonella typhimurium TA102 (Marzin and Phi, 1985). Concentrations of copper sulfate ranging from 10 to 3000 nM/plate (2.5 to 750 μg/plate) were reported to be non-mutagenic. The threshold toxic dose (i.e. the dose which decreased the bacterial background lawn) was reported to be greater than 3000 nM (750 μg/plate), although the actual concentration was not specified. The use of a metabolic activation system was not indicated in the report and no detailed results data were presented.
A negative result was similarly reported for copper sulfate in the Ames test, using Salmonella typhimurium TA98 and TA100 (Moriya et al., 1983). The test was conducted with and without metabolic activation. Substances were tested at concentrations up to 5000 μg/plate, unless cytotoxicity was evident at lower doses. Cytotoxic concentrations were not reported specifically for copper sulfate.
The EU RAR (2008) concludes that copper sulfate is negative in vitro in bacterial cell reverse mutation assays up to and including cytotoxic doses.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2019-12-03 to 2020-01-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across hypothesis is based on transformation of the target and source substances to common compounds (scenario 1 of the RAAF). The target substance copper monoglycinate sulfate and the source substances copper sulfate and copper bisglycinate consist of the Cu2+ cation and the respective anion. The amino acid glycine is constituent of both the target substance copper monoglycinate sulfate and the source substance copper bisglycinate.
It is generally accepted that the Cu2+ cation (as measure for dissolved copper species) is the determining factor for toxicity and ecotoxicity, but not sulfate or glycine.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance copper monoglycinate sulfate is a chelate-complex which consists of the divalent copper ion as centre-ion and glycine as ligand. The remaining sulfate group stabilizes the center ion within the complex.
Copper monoglycinate sulfate and the source substance copper sulfate are ionic and consist of the Cu2+ cation and the respective anions. It is generally accepted that the copper cation is the determining factor for toxicity and ecotoxicity. Therefore, this read-across approach is based on the assumption that the metal cation of both the target and the source substance, copper, is the relevant component for assessment of toxicity and ecotoxicity.
The anion of the target substance is the essential amino acid glycine and the sulfate anion. In the source substance, it is the sulfate anion. These anions are not considered as (eco)toxicologically relevant at the given concentrations.
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
4. DATA MATRIX
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his locus
- Metabolic activation:
- with and without
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks:
- Glycine, Ishidate et al. (1984)
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks:
- Glycine, Ishidate et al. (1984)
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks:
- Glycine, Ishidate et al. (1984)
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks:
- Glycine, Ishidate et al. (1984)
- Species / strain:
- S. typhimurium, other: TA92
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks:
- Glycine, Ishidate et al. (1984)
- Species / strain:
- S. typhimurium, other: TA94
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks:
- Glycine, Ishidate et al. (1984)
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other: The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other: The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Key result
- Species / strain:
- S. typhimurium TA 102
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other: The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other:
- Remarks:
- The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Key result
- Species / strain:
- S. typhimurium TA 100
- 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:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other: The positive control of strain TA1537 was slightly outside the historical data range. However, since the number of revertants was increased this was not considered to reduce the validity of the test result.
- Conclusions:
- Copper glycinate sulfate is not considered to be mutagenic in the bacterial reverse mutation assay.
- Executive summary:
A guideline study assessing the mutagenic potential of the source substance copper bisglycinate is available (2020). In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation were tested up to 5000 µg/plate (pre-incubation and plate method). There was no evidence of induced mutant colonies over background. The positive controls induced the appropriate responses in the corresponding strains. Thus, copper bisglycinate is not classified as mutagenic according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
The European Union Risk Assessment Report (2008) has evaluated data from several bacterial in vitro assays and concludes that the overwhelming weight of evidence indicates that copper sulfate is negative in vitro in bacterial cell reverse mutation assays, and in several other bacterial cell assays up to and including cytotoxic doses (1000 - ~3000 μg/plate). Consideration of the available in vitro mutagenicity data results in the conclusion that there is no requirement for copper sulfate to be classified as mutagenic according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) according to the EU RAR (2008).
Glycine was tested for mutagenic potential in the study by Ishidate et al. (1984) in a bacterial reverse mutation assay. 6 tester strains of Salmonella typhimurium (TA92, TA1535, TA100, TA1537, TA94 and TA98) were treated with the test material, both with and without metabolic activation. 6 doses were applied, and the maximum doses were 35000 µg/plate (crystal) and 33000 µg/plate (powder), which represent the highest non-cytotoxic dose used in the experiment. No significant increases in the frequency of revertant colonies were observed for any of the bacterial strains at any dose of glycine, either with or without metabolic activation. Thus, glycine was not mutagenic in the bacterial reverse mutation assay.
In conclusion, copper monoglycinate sulfate is not expected to be mutagenic in the bacterial reverse mutation assay.
Referenceopen allclose all
Table 1: Number of revertants per plate (mean of 3 plates), first experiment [1b]
|
|
TA 98 |
TA100 |
TA 102 |
TA 1535 |
TA1537 |
||||||||||||||
Conc. |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
demin water |
15 |
no |
15 |
no |
71 |
no |
73 |
no |
264 |
no |
275 |
no |
10 |
no |
18 |
no |
7 |
no |
9 |
no |
0* |
13 |
no |
16 |
no |
60 |
no |
69 |
no |
259 |
no |
277 |
no |
17 |
no |
13 |
no |
9 |
no |
8 |
no |
50 |
11 |
no |
13 |
no |
63 |
no |
61 |
no |
280 |
no |
275 |
no |
12 |
no |
13 |
no |
8 |
no |
6 |
no |
150 |
14 |
no |
11 |
no |
65 |
no |
75 |
no |
309 |
no |
309 |
no |
12 |
no |
11 |
no |
4 |
no |
7 |
no |
500 |
10 |
no |
9 |
no |
67 |
no |
61 |
no |
317 |
no |
312 |
no |
11 |
no |
11 |
no |
8 |
no |
8 |
no |
1500 |
11 |
no |
9 |
no |
91 |
no |
87 |
no |
293 |
no |
285 |
no |
9 |
no |
8 |
no |
7 |
no |
6 |
no |
5000 |
7 |
no |
11 |
no |
110 |
no |
121 |
no |
291 |
no |
293 |
no |
10 |
no |
11 |
no |
6 |
no |
7 |
no |
Positive control |
s.g. |
no |
98 |
no |
s.g. |
no |
s.g. |
no |
605 |
no |
s.g. |
no |
221 |
no |
175 |
no |
232 |
no |
168 |
no |
*solvent/vehicle control with DMSO
Table 1b: Number of revertants per plate (mean of 3 plates), first experiment [1d]
|
|
TA 1537 |
||
Conc. |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
demin water |
5 |
no |
5 |
no |
0* |
4 |
no |
6 |
no |
50 |
5 |
no |
5 |
no |
150 |
5 |
no |
5 |
no |
500 |
4 |
no |
4 |
no |
1500 |
4 |
no |
4 |
no |
5000 |
2 |
no |
2 |
no |
Positive control |
91 |
no |
149 |
no |
Table 2: Number of revertants per plate (mean of 3 plates), second experiment
|
|
TA 98 |
TA100 |
TA 102 |
TA 1535 |
TA1537 |
||||||||||||||
Conc. |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
— MA |
Cytotoxic Precipitates (yes/no) |
+ MA |
Cytotoxic Precipitates (yes/no) |
demin water |
20 |
no |
19 |
no |
91 |
no |
89 |
no |
371 |
no |
376 |
no |
10 |
no |
10 |
no |
5 |
no |
8 |
no |
0* |
18 |
no |
20 |
no |
81 |
no |
83 |
no |
355 |
no |
365 |
no |
9 |
no |
12 |
no |
6 |
no |
8 |
no |
78 |
15 |
no |
16 |
no |
61 |
no |
61 |
no |
371 |
no |
357 |
no |
7 |
no |
6 |
no |
6 |
no |
6 |
no |
156 |
15 |
no |
16 |
no |
69 |
no |
65 |
no |
365 |
no |
371 |
no |
8 |
no |
7 |
no |
7 |
no |
6 |
no |
313 |
17 |
no |
16 |
no |
80 |
no |
77 |
no |
363 |
no |
365 |
no |
7 |
no |
7 |
no |
5 |
no |
4 |
no |
1250 |
16 |
no |
16 |
no |
85 |
no |
83 |
no |
357 |
no |
352 |
no |
6 |
no |
5 |
no |
3 |
no |
3 |
no |
2500 |
15 |
no |
16 |
no |
57 |
no |
63 |
no |
304 |
no |
320 |
no |
3 |
no |
5 |
no |
0 |
no |
1 |
no |
5000 |
0 |
no |
0 |
no |
4 |
no |
2 |
no |
269 |
no |
275 |
no |
1 |
no |
1 |
no |
0 |
no |
0 |
no |
Positive control |
s.g. |
no |
111 |
no |
s.g. |
no |
s.g. |
no |
765 |
no |
872 |
no |
248 |
no |
167 |
no |
152 |
no |
160 |
no |
*solvent/vehicle control with water
Concurrent controls
DMSO Experiment 1b/Vehicle control
Strain |
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl.1 |
11 |
15 |
60 |
62 |
256 |
288 |
15 |
15 |
11 |
8 |
Repl.2 |
14 |
13 |
62 |
80 |
272 |
264 |
17 |
10 |
8 |
9 |
Repl.3 |
14 |
19 |
58 |
66 |
248 |
280 |
18 |
14 |
8 |
8 |
Mean |
13 |
16 |
60 |
69 |
259 |
277 |
17 |
13 |
9 |
8 |
sd |
1.7 |
3.1 |
2.0 |
9.5 |
12.2 |
12.2 |
1.5 |
2.6 |
1.7 |
0.6 |
Demin Water Experiment 1b/Vehicle control
Strain |
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl.1 |
15 |
18 |
62 |
70 |
280 |
9 |
17 |
8 |
9 |
9 |
Repl.2 |
13 |
10 |
78 |
72 |
248 |
272 |
9 |
17 |
8 |
10 |
Repl.3 |
16 |
17 |
72 |
76 |
264 |
272 |
12 |
19 |
6 |
8 |
Mean |
15 |
15 |
71 |
73 |
264 |
275 |
10 |
18 |
7 |
9 |
sd |
1.5 |
4.4 |
8.1 |
3.1 |
16.0 |
4.6 |
1.7 |
1.2 |
1.2 |
1.0 |
Positive control Experiment 1b
Strain |
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Substance |
NPD |
BaP |
Na-azide |
2-AA |
NPD |
2-AA |
Na-azide |
2-AA |
NPD |
2-AA |
Repl.1 |
s.g. |
100 |
s.g. |
s.g. |
576 |
s.g. |
220 |
180 |
232 |
160 |
Repl.2 |
s.g. |
80 |
s.g. |
s.g. |
608 |
s.g. |
228 |
172 |
240 |
192 |
Repl.3 |
s.g. |
114 |
s.g. |
s.g. |
632 |
s.g. |
216 |
172 |
224 |
152 |
Mean |
-- |
98 |
-- |
-- |
605 |
-- |
221 |
175 |
232 |
168 |
sd |
-- |
17.1 |
-- |
-- |
28.1 |
-- |
6.1 |
4.6 |
8.0 |
21.2 |
f(l) |
>2 |
6.13 |
>2 |
>2 |
2.34 |
>2 |
22.10 |
13.46 |
25.78 |
21.00 |
Rev. abs. |
-- |
82 |
-- |
-- |
346 |
-- |
211 |
162 |
223 |
160 |
DMSO Experiment 1d/Vehicle control
Strain |
TA1537 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
4 |
5 |
Repl. 2 |
3 |
7 |
Repl. 3 |
5 |
6 |
Mean |
5 |
6 |
SD |
1.0 |
1.0 |
Demin Water Experiment 1d/Vehicle control
Strain |
TA1537 |
|
Induction |
-S9 |
+S9 |
Repl. 1 |
4 |
6 |
Repl. 2 |
5 |
4 |
Repl. 3 |
6 |
5 |
Mean |
5 |
5 |
SD |
1.0 |
1.0 |
Positive control Experiment 1d
Strain |
TA1537 |
|
Induction |
-S9 |
+S9 |
Substance |
Na-Azide |
2-AA |
Repl. 1 |
96 |
160 |
Repl. 2 |
92 |
148 |
Repl. 3 |
84 |
140 |
Mean |
91 |
149 |
SD |
6.1 |
10.1 |
f(l) |
22.75 |
24.83 |
Rev. abs. |
87 |
143 |
s.g.= strong growth, too strong for counting of revertants
f(l) = increase factor
Rev.abs. = absolute revertants
Demin. Water Experiment 2/Vehicle control
Strain |
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl.1 |
19 |
20 |
96 |
96 |
352 |
368 |
9 |
10 |
6 |
9 |
Repl.2 |
20 |
19 |
92 |
92 |
384 |
384 |
12 |
10 |
5 |
7 |
Repl.3 |
20 |
19 |
84 |
80 |
376 |
376 |
10 |
11 |
4 |
7 |
Mean |
20 |
19 |
91 |
89 |
371 |
376 |
10 |
10 |
5 |
8 |
sd |
0.6 |
0.6 |
6.1 |
8.3 |
16.7 |
8.0 |
1.5 |
0.6 |
1.0 |
1.2 |
DMSO Experiment 2/Vehicle control
Strain |
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl.1 |
18 |
19 |
76 |
80 |
352 |
360 |
9 |
11 |
6 |
9 |
Repl.2 |
18 |
20 |
80 |
84 |
360 |
368 |
8 |
15 |
6 |
6 |
Repl.3 |
18 |
21 |
88 |
84 |
352 |
368 |
10 |
10 |
6 |
8 |
Mean |
18 |
20 |
81 |
83 |
355 |
365 |
9 |
12 |
6 |
8 |
sd |
0.0 |
1.0 |
6.1 |
2.3 |
4.6 |
4.6 |
1.0 |
2.6 |
0.0 |
1.5 |
Positive control Experiment 2
Strain |
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Substance |
NPD |
BaP |
Na-azide |
2-AA |
NPD |
2-AA |
Na-azide |
2-AA |
NPD |
2-AA |
Repl.1 |
s.g. |
112 |
s.g. |
s.g. |
752 |
856 |
240 |
164 |
156 |
168 |
Repl.2 |
s.g. |
112 |
s.g. |
s.g. |
768 |
864 |
264 |
168 |
132 |
168 |
Repl.3 |
s.g. |
108 |
s.g. |
s.g. |
776 |
896 |
240 |
168 |
168 |
144 |
Mean |
-- |
111 |
-- |
-- |
765 |
872 |
248 |
167 |
152 |
160 |
sd |
-- |
2.3 |
-- |
-- |
12.2 |
21.2 |
13.9 |
2.3 |
18.3 |
13.9 |
f(l) |
>2 |
5.55 |
>2 |
>2 |
2.15 |
2.39 |
24.80 |
13.92 |
25.33 |
20.00 |
Rev. abs. |
-- |
91 |
-- |
-- |
410 |
507 |
238 |
155 |
146 |
152 |
s.g.= strong growth, too strong for counting of revertants
f(l) = increase factor
Rev.abs. = absolute revertants
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
A guideline study assessing the mutagenic potential of the source substance copper bisglycinate is available (2020). In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation were tested up to 5000 µg/plate (pre-incubation and plate method). There was no evidence of induced mutant colonies over background. The positive controls induced the appropriate responses in the corresponding strains. Thus, copper bisglycinate is not classified as mutagenic according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
The European Union Risk Assessment Report (2008) has evaluated data from several bacterial in vitro assays and concludes that the overwhelming weight of evidence indicates that copper sulfate is negative in vitro in bacterial cell reverse mutation assays, and in several other bacterial cell assays up to and including cytotoxic doses (1000 - ~3000 μg/plate). Consideration of the available in vitro mutagenicity data results in the conclusion that there is no requirement for copper sulfate to be classified as mutagenic according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) according to the EU RAR (2008).
Glycine was tested for mutagenic potential in the study by Ishidate et al. (1984) in a bacterial reverse mutation assay. 6 tester strains of Salmonella typhimurium (TA92, TA1535, TA100, TA1537, TA94 and TA98) were treated with the test material, both with and without metabolic activation. 6 doses were applied, and the maximum doses were 35000 µg/plate (crystal) and 33000 µg/plate (powder), which represent the highest non-cytotoxic dose used in the experiment. No significant increases in the frequency of revertant colonies were observed for any of the bacterial strains at any dose of glycine, either with or without metabolic activation. Thus, glycine was not mutagenic in the bacterial reverse mutation assay.
In conclusion, copper monoglycinate sulfate is not expected to be mutagenic in the bacterial reverse mutation assay.
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