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EC number: 611-056-6 | CAS number: 538313-26-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
According to OECD Guideline 471 (Bacterial Reverse Mutation Assay), the mutagenicity of the test substance was judged to be negative because the numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all tester strains. Therefore, it is concluded that CuDABT has no ability to induce mutations under the present test conditions.
According to OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test), a genotoxic activity was observed in absence of metabolic activation, with both a short and a continuous treatment. In return, no genotoxic activity was observed in presence of metabolic activation.
According to tRegulation 1907/2008/EC, a mutagenic category 2 classification is proposed. Based on the discussion below in the "Justification for classification or non-classification" section and in accordance with Section 1 of Annex XI, a somatic cell in vivo study for CuDABT, as required under Section 8.4 of Annex VIII does not appear scientifically necessary.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- September 16, 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Only duplicates and no justification for it. Justification of solution stability is not tested adequately. Statistical processing was not performed.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Only duplicates plates were used instead of triplicates. Justification of solution stability is not tested adequately. Statistical processing was not performed.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 was prepared from the liver of rats. The S9 was stored below -80°C until use and thawed just before use. S9 mix (1 ml) = 8 μmol MgCl2+33 μmol KCl+5 µmol Glucose-6-phosphate+4 μmol NADPH+4 μmNADH+100 μmol sodiumphosphate buffer (pH 7.4)+0.1 mL S9
- Test concentrations with justification for top dose:
- The final numbers of prepared viable cells are (x 10^9 cells/mL):
TA100: dosage setting test=2.6 and main test=2.6
TA1535: dosage setting test=2.6 and main test=2.5
WP2uvrA: dosage setting test=4.1 and main test=4.0
TA98: dosage setting test=2.6 and main test=2.6
TA1537: dosage setting test=2.5 and main test=2.5 - Vehicle / solvent:
- DMSO (Dimethyl sulfoxide)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide 5AF-2), 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine·2HCl (ICR-191), 2-Aminoanthracene (2AA)
- Remarks:
- 2AA was the only one positive controle for all strains with S9 activation.
- Evaluation criteria:
- Cases where the number of reverse mutation colonies had increased by 2× or more the negative control value, and where dosage-dependency or reproducibility were seen in the increase, were treated as positive, and other cases as negative.
- Statistics:
- Statistical processing was not performed.
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
In the test results, the number of reverse mutation colonies was less than 2× that of the negative control value in all tested bacteria strains, regardless of the presence of the S9 mix. It was therefore determined that the mutagenicity was negative.
The number of reverse mutation colonies of the positive control was 2× or more that of the negative control, and the number of reverse mutation colonies of the negative control and positive control were within the range of the historical data. In addition, it was confirmed that the test system was free of contamination, and it was determined that the test was implemented normally.
From the above results, it was determined that CuDABT was free of mutagenicity under the test conditions. - Executive summary:
The ability of CuDABT to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). As a result, the mutagenicity of the test substance was judged to be negative because the numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all tester strains. Therefore, it is concluded that CuDABT has no ability to induce mutations under the present test conditions.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: chromosomal damage
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March to May 2016
- Reliability:
- 1 (reliable without restriction)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- yes
- Remarks:
- Highest concentration of test item lower than OECD recommandation but no impact on the study (positive result is obtained).
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- TEST ITEM: copper diammonium bitetrazole (CuDABT)
OTHER NAMES / CODE: Copper, diammine[5,5’-bi-1H-tetrazolato(2-)-kN1,kN1’] / CAS number: 538313-26-7 / EC number: 611-056-6 / Copper Diammine Bitetrazole
NAME ON IDENTIFICATION TAG: CuDABT
IPL REGISTRATION NUMBER: 160308
BATCH NUMBER: sponsor reference: P3656461; INERIS reference: 16AH596
EXPIRY DATE: 25/8/2020
APPEARANCE: blue powder
PURITY: 97.4%
SALT / BASE RATIO: unknown
WATER CONTENT: unknown
MOLECULAR WEIGHT: based on the formula [Cu(NH 3 ) 2 ](NO 3 ) 2 : 221,617 g/mol
CORRECTION FACTOR: 1.026*
QUANTITY SUPPLIED: 9.41 g
STORAGE CONDITIONS: room temperature (+20°±5°C), protected from light and humidity
STORAGE CONDITIONS: 5 years (i.e. up to expiry date 25/08/2020) - Species / strain / cell type:
- human lymphoblastoid cells (TK6)
- Details on mammalian cell type (if applicable):
- A stock of TK6 cells is preserved in liquid nitrogen at the laboratory. Cells were provided by ATCC (American Type Culture Collection). They have a stable caryotype (47, XY, 13+) and have a 16-18 hour doubling time.
Contamination by mycoplasma is checked using Mycoalert mycoplasma detection kit for each batch of the cells. Only the batches, which contain no mycoplasma, are used in the genotoxicity test. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction prepared using the metho describe by Ames (1975) in male rat OFA SD induced by Aroclor 1254 according to the standard operating procedures of the Institut Pasteur de Lille
- Test concentrations with justification for top dose:
- Assay without metabolic activation with 3-hour treatment with 24-hour recovery period (assay S9- 3h/+24h): 10.49, 13.11, 16.8, 20.48, 25.6, 32 and 40 µg/mL
Assay with metabolic activation with 3-hour treatment with 24-hour recovery period (assay S9+ 3h/+24h): 5.37, 6.71, 8.39, 10.49, 13.11, 16.38 and 20.48 µg/mL,
Assay without metabolic activation with 27-hour treatment without recovery period (assay S9- 27h/+0h): 1.76, 2.2, 2.75, 3.44, 4.29 and 5.37 µg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: As a previous study was performed with sterile water (CERI Study ref. K01-4798) while organic solvents did not allow a better formulation, sterile water was chosen as final solvent. - Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile water
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: griseofluvin
- Details on test system and experimental conditions:
- TEST SYSTEM: TK6 cell line is a continuous human lymphoblastoid cell line isolated from a culture of the spontaneous immortalised splenic lymphoblastes WIL2 which possesses a normal and stable p53 status (p53 +/+). This cell line is currently used in our lab and is one of the recommended cell lines in OECD guideline No. 487 (2014).
METHOD OF APPLICATION: in medium (RPMI 0 medium containing sodium bicarbonate, non-essential aminoacids, penicillin, streptomycin and HCl / RPMI10 medium containing 10% inactivated horse serum, pluronic acid, L-Glutamine, sodium pyruvate and Amphotericine B)
DOSE FORMULATION:
For the 1st toxicity assay (see also § 10.2), the test item copper diammonium bitetrazole (CuDABT) was suspended in sterile water (Fresenius, batch 13IMP031) at a maximum initial concentration of 20 mg/mL and used at 10% in culture medium, giving a final concentration of 2000 µg/mL. Successive dilutions were also prepared in sterile water and used at 10 % in culture medium.
For the 2nd toxicity assay (see also § 10.2), the test item copper diammonium bitetrazole (CuDABT) was also suspended in sterile water but at a maximum initial concentration of 1 mg/mL and used at 10% in culture medium, giving a final concentration of 100 µg/mL. Successive dilutions were also prepared in sterile water and used at 10 % in culture medium.
For the main assay, the test item copper diammonium bitetrazole (CuDABT) was suspended in sterile water at a maximum initial concentration of 10 mg/mL and successively diluted at 1 and 0.4 mg/mL. This formulation used at 10% in culture medium, gave a final concentration of 40 µg/mL. Successive dilutions using a dilution factor of 1.25 were also prepared in sterile water and used at 10 % in culture medium.
The stability of the test item in the solvent was unknown but preparations for treatment were performed just before use.
DURATION:
Without S9 mix: 3 h treatment + 24 h recovery (first et third experiment), 24 h treatment + 20 h recovery (second experiment, since the results of the first experiment were equivocal)
With S9 mix: 3 h treatment + 24 h recovery (both experiments)
NUMBER OF REPLICATIONS: duplicate
NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per concentration
DETERMINATION OF CYTOTOXICITY
- Method: population doubling (PD) is the log of the ratio of the final count at the time of harvesting (N) to the starting count (N0), divided by the log of 2. - Evaluation criteria:
- In the solvent control groups, the number of micronucleated cells per mononucleated were within the 95% control limits of the distribution of the laboratory’s historical negative control database, with however one minor exception.
Concurrently to the main assays, tests were carried out with reference mutagenic compounds (mitomycin C in the absence of metabolic activation and cyclophosphamide in the presence of metabolic activation via S9-mix) and with a reference aneugenic compound (griseofulvin in the continuous treatment in absence of metabolic activation), in order to demonstrate the sensitivity of the cells and the effectiveness of the metabolic activation system. Statistically significant increases in the number of micronucleated cells in the mononucleated cells were observed in the presence of mitomycin C, cyclophosphamide and griseofulvin. The values observed were consistent with historical data regarding extreme values already noted.
It is noteworthy that, in the treatment with metabolic activation, discrepant results were noted for the positive control cyclophosphamide with 15 and 43 micronucleates for cultures A and B, respectively. A second reading was thus performed on the second slide prepared for each of the 2 cultures. Discrepant results were noted for the positive control cyclophosphamide with 5 and 56 micronucleates for cultures A and B. On the other hand, it is noteworthy that these slides presented a dark coloration rendering the reading difficult as the cytoplasm was almost as dark as the nuclei. Under these conditions, it was decided to not take into account such inaccurate results.
The test item was toxic. In accordance with the recommendations of the OECD guideline No. 487, the highest concentration of the test item presented a RPD of 45+/-5% of solvent controls, i.e. 55 +/-5% of cytostasis, but only in the short-term treatment without metabolic activation.
The acceptance criteria for the results were thus considered as fulfilled. - Statistics:
- The statistical comparison was performed using the X2 test, in which p = 0.05 was used as the lowest level of significance.
- Key result
- Species / strain:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- 1. RESULTS FOR CYTOTOXIC ACTIVITY
A first preliminary toxicity assay was carried out with concentrations ranging from 31.25 to 2000 µg/mL corresponding to the highest concentration recommended by the OECD Guideline No. 487. However, due to a very high level of toxicity on this whole range of concentrations in the 3 treatment schedules, no maximum concentrations could be selected for the main assay. The toxicity assay was thus reiterated with a lower and large range of concentrations (i.e. from 100 to 0.10 µg/mL.
In the second preliminary toxicity assay using a 3-hour treatment without metabolic activation followed by a recovery period, a potent cytotoxicity was observed at the highest concentration tested of 100 µg/mL, with no cell growth. The immediately lower concentration of 50 µg/mL also induced a too strong level of cytotoxicity with a RPD of -58.1% corresponding to percentage of cytostasis of 158.1%.
With an acceptable level of cytostasis, i.e. 60.1% (and a RPD of 39.9%) the concentration of 25 µg/mL was expected to induce a level of toxicity comprised between 60 and 50%. However, given the steep toxicity curve for the main assay, a higher concentration was chosen, i.e. 40 µg/mL, and a large and narrowed range of concentrations was tested (i.e. dilution factor of 1.25).
In the corresponding genotoxicity assay, the 2 highest concentrations of 40 and 32 µg/mL induced a too high toxicity with RPDs of -87.5 and -29.4%, respectively corresponding to percentages of cytostasis of 187.5 and 129.4%, respectively, when compared to the respective solvent control. With an acceptable level of cytostasis, i.e. 57.9% (and a RPD of 42.1%) the concentration of 25.6 µg/mL was retained as the maximum concentration to be assessed for genotoxicity. The two lower concentrations of 20.48 and 16.38 µg/mL were also assessed for genotoxicity.
In the second preliminary toxicity assay using a 3-hour treatment with metabolic activation followed by a recovery period, a potent cytotoxicity was observed at the highest concentration tested of 100 µg/mL, with no cell growth. The 2 immediately lower concentrations of 50 and 25 µg/mL also induced a too strong level of cytotoxicity with RPDs of -245.3 and 0.4% corresponding to percentage of cytostasis of 345.3 and 99.6%, respectively. With a moderate level of cytostasis at 12.5 µg/mL, i.e. 24.1% (and a RPD of 75.9%), a concentration comprised between 25 and 12.5 µg/mL was expected to induce a level of toxicity comprised between 60 and 50%. For the main assay, the concentration of 20.48 µg/mL was chosen as the highest concentration, and a large and narrowed range of concentrations was tested.
In the corresponding genotoxicity assay, the highest concentration of 20.48 µg/mL induced only a slight toxicity with a RPD of 77.3% corresponding to a percentage of cytostasis of 22.7%, when compared to the respective solvent control. The concentration of 20.48 µg/mL was thus retained as the maximum concentration to be assessed for genotoxicity (see also §10.1). The two lower concentrations of 16.38 and 13.11 µg/mL were also assessed for genotoxicity.
In the second preliminary toxicity assay using a 27-hour treatment without metabolic activation without recovery period, a potent cytotoxicity was observed at the 4 highest concentrations tested ranging from 100 to 12.5 µg/mL, with no cell growth. The immediately lower concentration of 6.25 µg/mL still induced a too strong level of cytotoxicity with a RPD of –17.6% corresponding to percentage of cytostasis of 117.6%. With a strong but acceptable level of cytostasis, i.e. 39.3% (and a RPD of 60.7%) at the concentration of 3.13 µg/mL, a concentration comprised between 3.13 and 6.25 µg/mL was expected to induce a level of toxicity comprised between 60 and 50%. For the main assay, the concentration of 5.37 µg/mL was chosen as the highest concentration, and a large and narrowed range of concentrations was tested.
In the corresponding genotoxicity assay, the 2 highest concentrations of 5.37 and 4.29 µg/mL induced a too high toxicity with RPDs of 16.4 and 28%, respectively corresponding to percentages of cytostasis of 83.6 and 72%, when compared to the respective solvent control. With an acceptable level of cytostasis, i.e. 38.5% (and a RPD of 61.4%) the concentration of 3.44 µg/mL was retained as the maximum concentration to be assessed for genotoxicity (see also §10.1). The two lower concentrations of 2.75 and 2.20 µg/mL were also assessed for genotoxicity.
2. RESULTS FOR GENOTOXIC ACTIVITY
The investigation of a genotoxic activity of the test item copper diammonium bitetrazole (CuDABT) (sponsor reference: P3656461, INERIS reference: 16AH596) sponsored by AUTOLIV Romania S.r.1 (IRO) has been carried out compliance with the OECD Guideline 487 (2014), using the in vitro mammalian cell micronucleus test on TK6 lymphoblastoid human cells. The test was performed at the maximum concentration compatible with the toxic activity of the test item in the three experimental conditions with and without S9-mix, both with and without recovery period. Two concentrations below this were also analysed.
All the concentrations tested in this study are expressed as µg/mL of pure copper diammonium bitetrazole (CuDABT).
In the short-term treatment without metabolic activation followed by a recovery period (assay S9- 3h/+ 24h), the test item copper diammonium bitetrazole (CuDABT) induced a statistically and biologically significant increase in the number of micronucleated cells at the highest concentration analyzed of 25.6 µg/mL.
Indeed, a mean of 18.5 micronucleated mononucleated cells were observed per 1000 cells, vs. 1 in the negative control. This value was clearly over the 95% confidence interval of historical data for negative control (1.98 – 3.98), and clearly above the intervals of extreme values (1-5.8). Moreover, a dose-response, as evaluated via the ANOVA trend test was also noted. The test item copper diammonium bitetrazole (CuDABT) was thus considered as genotoxic under this condition.
Moreover, when using an extended time of exposure without metabolic activation, i.e. assay S9- 27h/+0h, the test item copper diammonium bitetrazole (CuDABT) induced a statistically and biologically significant increase in the number of micronucleated cells at the highest concentration analyzed of 3.44 µg/mL. Indeed, at this concentration, 9 micronucleated mononucleated cells were observed per 1000 cells, vs. 2 in the negative control. Moreover, the trend test (ANOVA) demonstrated a statistical significance, and this value was over the 95% confidence interval of historical data for negative control (1.66 – 2.93), and even above the interval of extreme values (1-6.8). Therefore, copper diammonium bitetrazole (CuDABT) was considered as genotoxic under this condition.
In the short-term treatment with metabolic activation followed by a recovery period (assay S9+ 3h/+ 24h), the test item copper diammonium bitetrazole (CuDABT) induced a statistically significant increase in the number of micronucleated cells at the intermediary concentration analyzed of 16.38 µg/mL. Indeed, 8 micronucleated mononucleated cells were observed per 1000 cells, vs. 3 in the negative control. The trend test (ANOVA) failed at demonstrating a statistically significance but this value was over the 95% confidence interval of historical data for negative control (2.42 – 4.04), and even above the interval of extreme values (1 – 6). However, it is noteworthy that discrepant results were noted between cultures for both highest concentrations. Therefore a second reading was performed.
The complementary reading exhibited homogenous results between the 2 cultures. Otherwise, no statistically significant increase in the number of micronucleated cells were observed at the concentration analyzed of 20.48 and 16.38 µg/mL. Indeed, 3 and 3.5 micronucleated mononucleated cells were observed per 1000 cells, vs. 1 in the negative control; theses values were inside the 95% confidence interval of historical data for negative control.
After compilation of the results of both readings (Table 10-c), a statistically significant increase in the number of micronucleated cells was still noted at the intermediary concentration analyzed of 16.38 µg/mL, with 5.5 micronucleated mononucleated cells observed per 1000 cells, vs. 2 in the negative control. This value was over the 95% confidence interval of historical data for negative control (2.42 – 4.04), but the trend test (ANOVA) failed at demonstrating a statistically significance and this value was inside the interval of extreme values for negative control (1 – 6).
The genotoxic activity of the test item copper diammonium bitetrazole (CuDABT) was thus considered as not genotoxic under this condition. - Conclusions:
- The genotoxic activity of the test item copper diammonium bitetrazole (CuDABT) (Sponsor batch reference: P3656461, INERIS batch reference: 16AH596) provided by AUTOLIV Romania S.r.1 (IRO) was assessed by means of the in vitro micronucleus test in TK6 lymphoblastoid human cells treated in presence of metabolic activation with a short-term treatment and in absence of metabolic activation, with both a short-term and with a continuous treatment according to OECD guideline (OECD 487, 2014).
The acceptance criteria for the assay were fulfilled. The study is thus considered as valid.
Under these experimental conditions, a genotoxic activity was observed in absence of metabolic activation, with both a short and a continuous treatment.
In return, no genotoxic activity was observed in presence of metabolic activation. - Executive summary:
AIM
The investigation of a genotoxic activity of the test item copper diammonium bitetrazole (CuDABT) (Sponsor batch reference: P3656461, INERIS batch reference: 16AH596) provided by AUTOLIV Romania S.r.1 (IRO) has been carried out compliance with the OECD Guideline No. 487 (September 2014), using in vitro mammalian cell micronucleus test on TK6 lymphoblastoid human cells.
METHODS
Cell strain : TK6 lymphoblastoid human cells
Culture medium : RPMI 1640
Solvent used : sterile water
Purity : 97.4% (taken into account in the calculations)
Stability in solvent : unknown (dilutions were prepared extemporaneously)
PRELIMINARY CYTOTOXICITY ASSAY
Carried out both without and with metabolic activation using Aroclor1254-induced S9 from rat livers.
Number of experimental conditions : 3
Number of cultures/concentration : 1
Factor limiting the maximum concentration analyzed : none, maximum concentration according to OECD guideline, i.e. 2000 µg/mL
Expression of the concentrations
used in the toxicity assay : µg/mL of pure copper diammonium bitetrazole (CuDABT)
It is noteworthy that a first preliminary toxicity assay was carried out at the concentrations ranging from 2000 to 31.25 µg/mL (see also § 10.2). However, due to a very high level of toxicity in the 3 treatment schedules, no range of concentrations could be chosen for the main assay. The toxicity assay was thus reiterated with a lower and large range of concentrations (i.e. from 100 to 0.10 µg/mL). The summary results are presented and commented hereafter.
Assay S9 - 3h/+24h Assay S9+ 3h/+24h Assay S9 - 27h/+0h Compound Conc. in Mg/mL Relative Population Doubling Cytostasis (%) Relative Population Doubling Cytostasis (%) Relative Population Doubling Cytostasis (%) Negative control 0 100 0 100 0 100 0 copper diammonium bitetrazole (CuDABT) 100 * * * * * * 50 -58.1 158.1 -245.3 345.3 * * 25 39.9 60.1 0.4 99.6 * * 12.5 82.8 17.2 75.9 24.1 * * 6.25 83.7 16.3 85.7 14.3 -17.6 117.6 3.13 91.2 8.8 91.1 8.9 60.7 39.3 1.56 85.3 14.7 108.0 -8.0 110.6 -10.6 0.78 73.2 26.8 94.1 5.9 111.0 -11.0 0.39 89.2 10.8 86.9 13.1 106.2 -6.2 0.20 88.9 11.1 83.3 16.7 113.6 -13.6 0.10 32.4 67.6 94.1 5.9 111.0 -11.0 *: Calculation could not be performed due to a high level of cytotoxicity In the reiterated preliminary toxicity assays using a 3-hour treatment without metabolic activation followed by a recovery period, a potent cytotoxicity was observed at the highest concentration tested of 100 µg/mL, with no cell growth. The immediately lower concentration of 50 µg/mL also induced a too strong level of cytotoxicity with a RPD of -58.1% corresponding to a percentage of cytostasis of 158.1%. With an acceptable level of cytostasis, i.e. 60.1% (and a RPD of 39.9%) the concentration of 25 µg/mL was expected to induce a level of toxicity comprised between 60 and 50%. However, given the steep toxicity curve for the main assay, a higher concentration was chosen, i.e. 40 µg/mL, and a large and narrowed range of concentrations was tested (i.e. dilution factor of 1.25).
In the reiterated preliminary toxicity assays using a 3-hour treatment with metabolic activation followed by a recovery period, a potent cytotoxicity was observed at the highest concentration tested of 100 µg/mL, with no cell growth. The 2 immediately lower concentrations of 50 and 25 µg/mL also induced a too strong level of cytotoxicity with RPDs of -245.3 and 0.4% corresponding to a percentage of cytostasis of 345.3 and 99.6%, respectively. With a moderate level of cytostasis at 12.5 µg/mL, i.e. 24.1% (and a RPD of 75.9%), a concentration comprised between 25 and 12.5 µg/mL was expected to induce a level of toxicity comprised between 60 and 50%. For the main assay, the concentration of 20.48 µg/mL was chosen as the highest concentration, and a large and narrowed range of concentrations was tested.
In the reiterated preliminary toxicity assays using a 27-hour treatment without metabolic activation without recovery period, a potent cytotoxicity was observed at the 4 highest concentrations tested ranging from 100 to 12.5 µg/mL, with no cell growth. The immediately lower concentration of 6.25 µg/mL still induced a too strong level of cytotoxicity with a RPD of –17.6% corresponding to a percentage of cytostasis of 117.6%. With a strong but acceptable level of cytostasis, i.e. 39.3% (and a RPD of 60.7%) at the concentration of 3.13 µg/mL, a concentration comprised between 3.13 and 6.25 µg/mL was expected to induce a level of toxicity comprised between 60 and 50%. For the main assay, the concentration of 5.37 µg/mL was chosen as the highest concentration, and a large and narrowed range of concentrations was tested.
GENOTOXICITY ASSAY
Carried out both without and with metabolic activation using Aroclor1254-induced S9 from rat livers.
Number of experimental conditions : 3
Number of cultures/concentration : 2
Number of analyzed cells : 2000 mononucleated cells / concentration
Factor limiting the maximum
concentration analyzed : cytotoxicity
Expression of the concentrations
used in the genotoxicity assays : µg/mL of pure copper diammonium bitetrazole (CuDABT)
Assay with 3 h treatment and 24 h recovery period:
- Without S9-mix : 40 – 32 – 25.6 – 20.48 – 16.38 – 13.11 – 10.49
- With S9-mix (5% S9-mix) : 20.48 – 16.38 – 13.11 – 10.49 – 8.39 – 6.71 – 5.37
Assay with 27 h treatment and no recovery period:
- Without S9-mix : 5.37 – 4.29 – 3.44 – 2.75 – 2.20 – 1.76
In bold, concentrations actually assessed
Positive controls
- Short-term treatment
Without S9-mix : mitomycin C 0.5 µg/mL
With S9-mix : cyclophosphamide 10 µg/mL
- Continuous treatment : mitomycin C 0.1 µg/mL
griseofulvin 10 µg/mL
RESULTS:
The test item copper diammonium bitetrazole (CuDABT) induced biologically significant increases in the number micronucleated cells in both short- and long-term treatments without metabolic activation. In return, after a short-term treatment with metabolic activation, the test item induced no statistically or biologically significant increases in the number of micronucleated cells. The copper diammonium bitetrazole (CuDABT) was thus considered as genotoxic in absence of metabolic activation.
Genotoxic activity study using in vitro micronucleus test on TK6 cells Conc. in pg/mL Relative Population Doubling (%) Cytostasis (%) Mean number of micronucleated cells per 1000 mononucleated cells P ANOVA S9- 3h/+24h Negative control 0 100 0 1 - - Mitomycin C 0.5 36.1 63.9 54 <0.001 <0.001 copper diammonium bitetrazole (CuDABT) 25.6 42.1 57.9 18.5 <0.001 20.48 71.2 28.8 3 N.S. 16.38 88.4 11.6 2 N.S. S9+ 3h/+24h Negative control 0 100 0 2 - - Cyclophosphamide 10 44.4 55.6 49.5 <0.001 - copper diammonium bitetrazole (CuDABT) 20.48 77.3 22.7 4 N.S. N.S. 16.38 78.5 21.5 5.5 <0.05 13.11 90.6 9.4 3 N.S. S9- 27h/+0h Negative control 0 100 0 2 - - Mitomycin C 0.1 29.5 70.5 46.5 <0.001 - Griseofulvin 10 35.4 64.6 39.5 <0.001 - copper diammonium bitetrazole (CuDABT) 3.44 61.5 38.5 9 <0.01 <0.01 2.75 77.7 22.3 4.5 N.S. 2.2 85.5 14.5 3 N.S. Chi2test for number of micronucleates
N.S.=not statistically significant at the threshold of p< 0.05
CONCLUSION:
The genotoxic activity of the test item copper diammonium bitetrazole (CuDABT) (Sponsor batch reference: P3656461, INERIS batch reference: 16AH596) provided by AUTOLIV Romania S.r.1 (IRO) was assessed by means of the in vitro micronucleus test in TK6 lymphoblastoid human cells treated in presence of metabolic activation with a short-term treatment and in absence of metabolic activation, with both a short-term and with a continuous treatment according to OECD guideline (OECD 487, 2014).
The acceptance criteria for the assay were fulfilled. The study is thus considered as valid.
Under these experimental conditions, a genotoxic activity was observed in absence of metabolic activation, with both a short and a continuous treatment.
In return, no genotoxic activity was observed in presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- an in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro micronucleus study
Referenceopen allclose all
Test Results Table (Dosage Setting Test)
Test Implementation Period |
20/8/2010~23/8/2010 |
|||||||||||
Presence of Metabolically Active System |
Tested Substance Dose (μg/plate) |
No. of Reverse Mutations (No. of Colonies/Plate) |
||||||||||
Base Substitution Type |
Frame Shift Type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
-S9 mix |
Negative Controls |
91 |
7 |
21 |
19 |
13 |
||||||
99 (100) |
8 (8) |
23 (20) |
18 (19) |
14 (11) |
||||||||
110 |
8 |
15 |
20 |
7 |
||||||||
4.88 |
123 |
(116) |
12 |
(11) |
17 |
(22) |
18 |
(16) |
10 |
(11) |
||
108 |
10 |
27 |
13 |
11 |
||||||||
19.5 |
99 |
(110) |
8 |
(8) |
19 |
(20) |
14 |
(13) |
12 |
(12) |
||
121 |
8 |
21 |
11 |
12 |
||||||||
78.1 |
115 |
(106) |
6 |
(9) |
28 |
(23) |
13 |
(11) |
10 |
(7) |
||
97 |
11 |
17 |
8 |
4 |
||||||||
313 |
129 |
(113) |
8 |
(6) |
20 |
(21) |
15 |
(17) |
6 |
(7) |
||
97 |
4 |
22 |
18 |
7 |
||||||||
1250 |
142 |
(128) |
5 |
(6) |
11 |
(11) |
13 |
(14) |
3 |
(6) |
||
114 |
7 |
10 |
14 |
6 |
||||||||
5000 |
154 |
(135) |
4 |
(3) |
12 |
(9) |
13 |
(10) |
6 |
(4) |
||
115 |
2 |
6 |
7 |
2 |
||||||||
+S9 mix |
Negative Controls |
115 |
(102) |
6 |
(9) |
18 |
(22) |
26 |
(23) |
8 |
(12) |
|
108 |
8 |
20 |
23 |
13 |
||||||||
83 |
13 |
27 |
21 |
15 |
||||||||
4.88 |
110 |
(126) |
13 |
(9) |
15 |
(22) |
30 |
(28) |
13 |
(13) |
||
142 |
10 |
28 |
25 |
12 |
||||||||
19.5 |
136 |
(135) |
7 |
(9) |
21 |
(22) |
30 |
(30) |
13 |
(13) |
||
133 |
10 |
23 |
25 |
12 |
||||||||
78.1 |
118 |
(126) |
13 |
(10) |
19 |
(22) |
30 |
(30) |
12 |
(12) |
||
134 |
7 |
25 |
29 |
8 |
||||||||
313 |
104 |
(110) |
11 |
(11) |
15 |
(11) |
11 |
(16) |
12 |
(14) |
||
115 |
11 |
7 |
20 |
16 |
||||||||
1250 |
98 |
(102) |
8 |
(8) |
13 |
(12) |
14 |
(11) |
11 |
(9) |
||
105 |
8 |
10 |
8 |
7 |
||||||||
5000 |
114 |
(135) |
12 |
(12) |
22 |
(19) |
5 |
(8) |
7 |
(7) |
||
156 |
12 |
16 |
11 |
7 |
||||||||
Positive Controls |
Those not requiring S9 mix |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
|||||
Dose (μg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
|||||||
No. of Colonies/Plate |
709 |
(705) |
275 |
(269) |
363 |
(373) |
386 |
(386) |
1899 |
(1886) |
||
701 |
262 |
382 |
386 |
1872 |
||||||||
Those requiring S9 mix |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
||||||
Dose (μg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|||||||
No. of Colonies/Plate |
1034 |
(1011) |
261 |
(274) |
502 |
(521) |
341 |
(337) |
428 |
(419) |
||
988 |
286 |
539 |
332 |
409 |
||||||||
Test Results Table (The Test)
Test Implementation Period |
27/8/2010~30/8/2010 |
|||||||||||
Presence of Metabolically Active System |
Tested Substance Dose (μg/plate) |
No. of Reverse Mutations (No. of Colonies/Plate) |
||||||||||
Base Substitution Type |
Frame Shift Type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
-S9 mix |
Negative Controls |
99 |
15 |
26 |
27 |
6 |
||||||
104 (104) |
8 (10) |
23 (26) |
15 (20) |
7 (8) |
||||||||
110 |
8 |
28 |
19 |
11 |
||||||||
313 |
115 |
(123) |
7 |
(9) |
21 |
(19) |
19 |
(16) |
10 |
(9) |
||
130 |
11 |
16 |
12 |
7 |
||||||||
625 |
109 |
(110) |
5 |
(8) |
27 |
(20) |
21 |
(13) |
4 |
(12) |
||
133 |
15 |
18 |
13 |
4 |
||||||||
1250 |
126 |
(137) |
14 |
(13) |
21 |
(23) |
14 |
(14) |
5 |
(6) |
||
147 |
12 |
25 |
14 |
7 |
||||||||
2500 |
114 |
(110) |
10 |
(8) |
19 |
(17) |
18 |
(19) |
6 |
(5) |
||
105 |
5 |
14 |
20 |
4 |
||||||||
5000 |
142 |
(132) |
11 |
(13) |
16 |
(14) |
11 |
(10) |
8 |
(5) |
||
122 |
14 |
11 |
9 |
2 |
||||||||
+S9 mi |
Negative Controls |
115 |
(117) |
10 |
(8) |
35 |
(33) |
16 |
(22) |
16 |
(21) |
|
121 |
5 |
28 |
23 |
22 |
||||||||
116 |
8 |
36 |
27 |
25 |
||||||||
313 |
120 |
(108) |
3 |
(8) |
26 |
(21) |
27 |
(27) |
12 |
(16) |
||
96 |
13 |
16 |
27 |
19 |
||||||||
625 |
114 |
(128) |
15 |
(12) |
18 |
(22) |
26 |
(26) |
21 |
(17) |
||
142 |
9 |
25 |
26 |
13 |
||||||||
1250 |
98 |
(110) |
12 |
(14) |
28 |
(25) |
18 |
(19) |
14 |
(17) |
||
122 |
15 |
21 |
19 |
20 |
||||||||
2500 |
153 |
(138) |
6 |
(9) |
19 |
(21) |
25 |
(24) |
5 |
(6) |
||
123 |
11 |
22 |
22 |
6 |
||||||||
5000 |
105 |
(112) |
8 |
(11) |
22 |
(21) |
14 |
(15) |
4 |
(5) |
||
118 |
14 |
19 |
15 |
6 |
||||||||
Positive Controls |
Those not requiring S9 mix |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
|||||
Dose (μg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
|||||||
No. of Colonies/Plate |
690 |
(687) |
286 |
(265) |
373 |
(380) |
384 |
(393) |
1521 |
(1542) |
||
684 |
244 |
386 |
401 |
1563 |
||||||||
Those requiring S9 mix |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
||||||
Dose (μg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|||||||
No. of Colonies/Plate |
941 |
(1000) |
293 |
(301) |
555 |
(552) |
280 |
(305) |
523 |
(446) |
||
1059 |
308 |
548 |
329 |
368 |
||||||||
| Genotoxic activity study using in vitro micronucleus test on TK6 cells | ||||||
| Conc. in pg/mL | Relative Population Doubling (%) | Cytostasis (%) | Mean number of micronucleated cells per 1000 mononucleated cells | P | ANOVA | |
| S9- 3h/+24h | ||||||
| Negative control | 0 | 100 | 0 | 1 | - | - |
| Mitomycin C | 0.5 | 36.1 | 63.9 | 54 | <0.001 | <0.001 |
| copper diammonium bitetrazole (CuDABT) | 25.6 | 42.1 | 57.9 | 18.5 | <0.001 | |
| 20.48 | 71.2 | 28.8 | 3 | N.S. | ||
| 16.38 | 88.4 | 11.6 | 2 | N.S. | ||
| S9+ 3h/+24h | ||||||
| Negative control | 0 | 100 | 0 | 2 | - | - |
| Cyclophosphamide | 10 | 44.4 | 55.6 | 49.5 | <0.001 | - |
| copper diammonium bitetrazole (CuDABT) | 20.48 | 77.3 | 22.7 | 4 | N.S. | N.S. |
| 16.38 | 78.5 | 21.5 | 5.5 | <0.05 | ||
| 13.11 | 90.6 | 9.4 | 3 | N.S. | ||
| S9- 27h/+0h | ||||||
| Negative control | 0 | 100 | 0 | 2 | - | - |
| Mitomycin C | 0.1 | 29.5 | 70.5 | 46.5 | <0.001 | - |
| Griseofulvin | 10 | 35.4 | 64.6 | 39.5 | <0.001 | - |
| copper diammonium bitetrazole (CuDABT) | 3.44 | 61.5 | 38.5 | 9 | <0.01 | <0.01 |
| 2.75 | 77.7 | 22.3 | 4.5 | N.S. | ||
| 2.2 | 85.5 | 14.5 | 3 | N.S. | ||
Chi2test for number of micronucleates
N.S.=not statistically significant at the threshold of p< 0.05
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
According to OECD Guideline 471 (Bacterial Reverse Mutation Assay), the mutagenicity of the test substance was judged to be negative because the numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all tester strains. Therefore, it is concluded that CuDABT has no ability to induce mutations under the present test conditions.
According to OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test), a genotoxic activity was observed in absence of metabolic activation, with both a short and a continuous treatment. In return, no genotoxic activity was observed in presence of metabolic activation.
The R7a guidance states that for an in vivo genotoxicity proposal to be included in the dossier:
“In REACH Annex VIII, following a positive result in an in vitro mammalian cell mutagenicity test, adequately conducted somatic cell in vivo testing is required to ascertain if this potential can be expressed in vivo. In cases where it can be sufficiently deduced that a positive in vitro finding is not relevant for in vivo situations (e.g. due to the effect of the test substances on pH or cell viability, in vitro-specific metabolism: see
also Section R.7.7.4.1), or where a clear threshold mechanism coming into play only at high concentrations that will not be reached in vivo has been identified (e.g. damage to non-DNA targets at high concentrations), in vivo testing will not be necessary.”
Section R.7.7.4.1 also states:
“In a more formal approach, documentation can include reference to a related substance or group of substances that leads to the conclusion of concern or lack of concern. This can either be presented according to scientific logic (read-across) or sometimes as a mathematical relationship of chemical similarity.”
Below is comparison of the substance properties of Copper, diammine[5,5’-bi-1Htetrazolato(2-)-kN1, kN1’] (CuDABT) to Copper (2+), bis [N-{ amino (imino-KN) methyl} urea-KO]-, nitrate (1:2) (CuGUN):
Property / Classification |
CuDABT |
CuGUN |
Melting |
293°C |
225°C |
Boiling |
293°C |
225°C |
Relative Density |
2.222 |
1.919 |
Vapor pressure |
1.27x10^-8 |
0.9x10^-7 |
Kow |
- 3.39 |
0.3 |
Water solubility |
< 0.01 g/L |
< 0.01 g/L |
Acute toxicity - oral: |
Not classified |
Acute Tox. 4 |
Skin corrosion / irritation: |
Not classified |
Skin Irrit. 2 |
Serious damage / eye irritation |
Not classified |
Eye Damage 1 |
Skin sensitisation: |
Skin Sens. 1B |
Skin Sens. 1B |
Germ cell mutagenicity: |
Muta. 2 |
Muta. 2 |
Specific target organ toxicity – repeated exposure: |
Not classified |
STOT Rep. Exp. 2 |
The two substances demonstrate the same physico-chemical properties. The two substances have low/no acute toxicity and skin sensitization.
The Ames test is negative for both substances and they both demonstrate a positive result in vitro:
- An OECD guidelines 487 for CuDABT resulted in negative genotoxic activity in presence of metabolic activation and low positive result in absence of metabolic activation CuDABT (5 times below the positive control)
- An OECD guidelines 476, with mouse lymphoma L5178Y TK+/- for CuGUN showed mutagenic activity in the presence metabolizing system and inconclusive results in absence of metabolizing system.
According to 1907/2006 (REACh) regulation, as the In Vitro Mammalian Cell Gene Mutation Test (476) was positive for CuGUN, an in vivo genetic toxicity assay was performed : In Vivo Mammalian Cell Gene Mutation Assay (OECD guidelines 489) on male rats treated by oral gavage with CuGUN (at 200, 400 and 800 mg/kg/day (two daily treatments at 24 hour intervals or X2)).
Under the experimental conditions, CuGUN did not induce statistically or biologically significant increases in DNA strand breaks at all tested concentrations in male rat isolated glandular stomach cells after oral administration. Therefore, CuGUN does not have genotoxic activity in this organ. Under these same experimental conditions, CuGUN induced dose-related statistically and biologically significant increases in DNA strand breaks at 400 and 800 mg/kg/day (x2) in male rat isolated liver cells after oral administration. Therefore, CuGUN produces genotoxic activity in this organ. Notably the magnitude of the observed result was 4.6-fold below the results for the methylmethane sulfonate positive control.
According to the R7.a guidance,. the low solubility and precipitation of a substance in in vitro mammalian cell assays has to be considered to assess the relevance of the test to the conditions in vivo.
The additional in vivo testing (OECD 476) conducted with CuGUN resulted in ambiguous results due to the low solubility of the substance, and did not allow definition of a clear dose-effect relationship.
By comparison of the whole data sets and the physico-chemical/toxicological profiles of the two substances, an in vivo genetic toxicity assay performed on CuDABT will present the same difficulties of interpretation. This test will allow the categorization of CuDABT as a mutagen Category 2. In fact, the guidance on the application of the CLP criteria states the parameters to consider in the genotoxic classification of the substance are as follows:
Category 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.
The classification in Category 2 is based on:
– Positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:
– Somatic cell mutagenicity tests in vivo, in mammals; or
– Other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.
In contrast the classification as Category 1B is based on:
– positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; or – positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells. It is possible to derive this supporting evidence from mutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells; or – positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.
Therefore, for CuDABT the following must be considered:
- the use of the criteria described above to classify CuDABT as mutagen category 2 or 1B, - the REACH regulation annex VIII column II that states : “In REACH Annex VIII, following a positive result in an in vitro mammalian cell mutagenicity test, requires an adequately conducted somatic cell in vivo”
For CuDABT it has been established that:
- it is difficult to test CuDABT in vivo (due to solubility and related issues) and to establish a dose-effect relationship
- a classification of CuDABT as a mutagen Category 2 has been already proposed/applied for the substance
- an additional somatic cell in vivo result will not be a source of additional information to re-categorize the substance.
Based on the discussion above and in accordance with Section 1 of Annex XI, a somatic cell in vivo study for CuDABT, as required under Section 8.4 of Annex VIII does not appear scientifically necessary.
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.
