Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
There is no data available for the registered substance on genetic toxicity. However, there is data available for the source substances Fe(Na)EDDHA and zinc salts.
Salmonella and E. coli tests in vitro (Ames Tests):
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9),S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537, E. coli WP2 uvr A, OECD Guideline 471, GLP, +/-S9, negative
Zinc
ZnCl2, Ames Test, S. typhimurium strains TA102 and TA97, +/-S9 negative (Fujita, 1988)
ZnAc, Ames Test, S. typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98, +/-S9 negative (Thompson, 1989)
In-vitro Mammalian Chromosome Aberation Test
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9), Chinese hamster ovary cells (CCL61), OECD Guideline 473, GLP, +/-S9, negative
Zinc
ZnCl2, human lymphocytes -S9 positive (Deknudt and Deminatti, 1978)
ZnAc, Chinese hamster ovary cells, +/-S9 positive (Thompson, 1989)
In-vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay):
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9), L5178Y mouse lymphoma cells, OECD Guideline 476, GLP, +/-S9, negative
Zinc
ZnCl2, L5178Y mouse lymphoma cells, -S9 negative (Amacher and Paillet, 1980)
ZnAc, L5178Y mouse lymphoma cells, +/-S9 positive (Thompson, 1989)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- zinc acetate
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- zinc acetate
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- zinc acetate
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- zinc acetate
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 1538
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- The results of the Salmonella/mammalian microsome plate-incorporation assay for zinc acetate were uniformly negative. Zinc acetate was neither toxic (determined by reduction of bacterial lawn) nor mutagenic to any of the 5 strains tested over a dose range of 50-7200 µg/plate.
- Executive summary:
Ames test was performed with zinc acetate. The plate-incorporation assay was performed with Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The results of the Salmonella/mammalian microsome plate-incorporation assay for zinc acetate were uniformly negative. Zinc acetate was neither toxic (determined by reduction of bacterial lawn) nor mutagenic to any of the 5 strains tested over a dose range of 50-7200 µg/plate.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
Six concentrations ranging from 20.6 - 5000 µg/plate were tested with strain Salmonella typhimurium TA100 and strain Escherichia coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. Although the test substance was applied as a fine homogeneous suspension, no precipitates were detectable on the plates. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and 5000 µg/plate without metabolic activation.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria. - Conclusions:
- Based on the results of these experiment and on standard evaluation criteria, it is concluded that the test substance and its metabolites did not induce gene mutations in the strains of S.thyphimurium and E.coli used.
- Executive summary:
FeNaEDDHA was tested for mutagenic effects in vitro in histidine-required strains of Salmonella typhimurium and in a tryptophan-required strain of Escherichia coli. The following strains were used: S. thyphimurium TA 98, TA100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in bidistilled water and testen at 5 concentrations in the range of 312.5 - 5000 µg/plate in the presence and absence of metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 312.5 - 5000 µg/plate. The concentration of 5000 µg/plate represents the test limit dose. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as a positive control.
In both experiments, performed with and without metabolic activation, none of the tested concentrations of the test substance FeNaEDDHA led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Due to the presence of precipitates of the test substance on the microscopic slides, concentrations higher than 31.25 ug/ml (without metabolic activation) or 125 ug/ml (with metabolic activation) could not be scored. - Conclusions:
- It was concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with FeNaEDDHA.
- Executive summary:
FeNaEDDHA was investigated for clastogenic (chromosome-damaging) effects on Chinese hamster ovary cells in vitro in a GLP compliant study according to OECD Guideline 473 with and without extrinsic metabolic activation (S9). The test compound FeNaEDDHA was dissolved in DMSO and tested at each of the following conditions:
Experiments without metabolic activation:
- 18 hours treatment time:
original experiment: 7.81, 15.63 and 31.25 µg/mL
confirmatory experiment: 7.81, 15.63 and 31.25 µg/mL
- 42 hours treatment time: 7.81, 15.63 and 31.25 µg/mL
Final concentrations higher than 31.25 ug/mL of culture medium could not be scored due to solubility limitations. Mitomycin C (0.2 ug/mL) was used as a positive control in the 18 hours experiments.
Experiments with metabolic activation:
- 3 hours treatment followed by 15 hours recovery period:
original experiment: 31.25, 62.5 and 125 µg/mL
confirmatory experiment: 31.25, 62.5 and 125 µg/mL
- 3 hours treatment followed by 39 hours recovery, period: 31.25, 62.5 and 125 µg/mL
Final concentrations higher than 125 ug/mL of culture medium could not be scored due to solubility limitations. Cyclophosphamide (20.0 µg/mL) was used as a positive control in the 3 hours/15 hours experiments.
In addition, DNA distribution of cultures treated under the above described conditions (18 hours only) was determined by flow cytometry. These measurements allow to analyse the influence of the test substance on the cell cycle of CHO cells. In both the experiments performed without and with metabolic activation no significant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed. Flow cytometry experiments did not reveal any evidence for cell cycle disturbing activities of FeNaEDDHA either in the absence or in the presence of metabolic activation.
It was concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with FeNaEDDHA.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- please refer to field 'Any other information on results'
- Conclusions:
- Based on the result of two indipendently performed experiments and under the given experimental conditions, it is concluded that FeNaEDDHA and its metabolites did not show any mutagenic activity at the tk locus of mouse lymphoma L5178Y cells in the presence and absence of metabolic activation. The same result is expected for the target substance since it has the same constituents that act via the same mechanism as the source substance. No differences in the magnitude of the effects are expected.
- Executive summary:
FeNaEDDHA was tested for its ability to induce mutations at the tk locus (5-trifluoro-thymidine resistance) in L5178Y mouse lymphoma cells. The study consisted of a preliminary cytotoxicity range-finder and two independent experiments, each performed with and without metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial supernatant (S9 fraction).
In a first range-finder experiment the concentrations applied ranged from 0.49 to 1000.0 µg/mL separated by 2-fold intervals. Based on the results of the range finding study, 5 concentrations were chosen for the first mutagenicity experiment, separated by 4-fold intervals and ranging from 0.49 to 125.0 µg/mL in the part with and from 3.91 to 1000.0 µg/mL in the part without metabolic activation. At the highest concentrations the zero hour survival was 49.33% and 62.01% in the presence and absence of metabolic activation. In the confirmatory experiment, in the part with metabolic activation, the concentration range was increased ranging from 0.98 to 250.0 µg/mL. Without metabolic activation the same concentration range was used. With metabolic activation the relative survival at the highest concentration was 47.45%, without metabolic activation the value found was 81.90%. Negative (vehicle) and positive control treatments were included in each experiment with and without metabolic activation. The mutant frequencies of the negative controls were within normal ranges and the positive controls N-Nitrosodimethylamine (DMN, with metabolic activation) and Ethylmethansulfonate (EMS, without metabolic activation) produced statistically significant increases of mutant frequency. In the part performed with and without metabolic activation, no relevant increases in mutant frequency were observed after treatment with FeNaEDDHA.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- zinc acetate
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Dose-dependent positive responses to zinc acetate were obtained in the presence and absence of the S9 activation system, although the S9 reduced both the clastogenic response and the toxicity. These results indicate that zinc is an effective clastogen when presented to a susceptible cell population in an appropriate form. However, in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.
- Executive summary:
A chromosome aberration assay with zinc acetate was conducted in Chinese Hamster Ovary cells. Zinc acetate induced chromosome aberraion in the presence and absence of metabolic activation. These results indicate that zinc is an effective clastogen when presented to a susceptible cell population in an appropriate form.
However, in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- The results presented here indicate zinc is an effective mutagen when presented to a mouse lymphoma cells in an appropriate form. However, controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.
- Executive summary:
A mouse lymphoma TK+/- assay was perormed with zinc acetate. The results indicate zinc is an effective mutagen when presented to a mouse lymphoma cells in an appropriate form. However, controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Conclusions:
- Zinc chloride was not genotoxic in L5178Y mouse lymphoma cells.
- Executive summary:
Zinc chloride was tested for the potential to induce trifluorothymidine-resistant (TFT Res) mutants in L5178Y/TK+/- mouse lymphoma cell by directly exposing cells to varied doses for 3 h. 48 h after treatment, metal-treated cells and solvent controls were cloned in soft-agar media and plated. Trifluorothymidine resistance (TFT Res) was determined by adding 4 µg/ml TFT to one set of plates. All colonies growing either in the presence of TFT (TFT Res) or its absence (viable count colonies) were counted on day 7 after incubation at 37°C. Those TFT Res colonies which were equivalent in size to colonies growing in the solvent control viable count plates i.e., large, were scored as mutants. The result was negative for ZnCl2.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- lymphocytes: human
- Remarks:
- zinc chloride
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Conclusions:
- The results suggest that high levels of zinc (3 x 10E-3 M) are cytotoxic and that lower concentration (3 x 10E-5 M) can cause severe chromosome aberrations (dicentrics). It must be pointed out, however, that the concentration of zinc used in the present experiments are extremely high representing up to 1000 times the respective concentrations reported in the blood of people professionally contaminated by heavy metals and which have been shown to be 22.4 µg% for zinc.
- Executive summary:
Chromosome aberrations in human lymphocytes induced by zinc chloride were analysed. The results suggest that high levels of zinc (3 x 10E-3 M) are cytotoxic and that lower concentration (3 x 10E-5 M) can cause severe chromosome aberrations (dicentrics). It must be pointed out, however, that the concentration of zinc used in the present experiments are extremely high representing up to 1000 times the respective concentrations reported in the blood of people professionally contaminated by heavy metals and which have been shown to be 22.4 µg% for zinc.
Due to the fact that the Zn ion is a constituent of the target substance, the result is relevant for the target substance.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- not specified
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- not specified
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- not specified
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Conclusions:
- Zinc sulphate was negative in two tester strains Salmonella typhimurium TA97 and TA102.
- Executive summary:
Mutagenicity zinc sulphate was examined in Ames' tester strains, Salmonella typhimurium TA97 and TA102. The mutation test was carried out by the preincubation procedure described by Ames et al. The test chemicals were preincubated with S9 mix or phosphate buffer (pH7.4) for 20 min. Zinc sulphate was negative in two tester strains.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
Referenceopen allclose all
The results of the Salmonella/mammalian microsome plate-incorporation assay for zinc acetate were uniformly negative. Zinc acetate was neither toxic (determined by reduction of bacterial lawn) nor mutagenic to any of the 5 strains tested over a dose range of 50-7200 µg/plate.
Analytical control:
To confirm that the cells were actually exposed to the intened test concentrations and to confirm the stability of the test substance in the vehicle used, determination of the concentration of the test substance in solution was performed by UV/VIS-spectroscopy. The values found by analysis of the different samples were in agreement with the intended concentrations (between 90.2 and 96.1 %), thus demonstrating a sufficient stability of the test substance in the vehicle.
There were no known or occurrences in this study that were considered to have affected the quiality or integrity of the test data.
Table 1: Original experiment
With(+) or without(-) S9 Mix |
Test substance concentration (µg/plate) |
Number of revertants (number of colonies/plate) |
|||||
Base-pair substitution type |
Frameshift type |
||||||
TA 100 |
TA 1535 |
UP2 uvrA |
TA 102 |
TA 98 |
TA 1537 |
||
S9 Mix (+) |
Solvent control |
173 183 185 (180) |
33 16 21 (23) |
36 33 36 (35) |
357 360 282 (333) |
53 40 51 (48) |
8 16 7 (10) |
312.50 |
185 197 187 (190) |
19 18 25 (21) |
40 41 43 (41) |
330 331 321 (327) |
48 41 52 (47) |
7 8 8 (8) |
|
625.00 |
181 195 157 (178) |
21 20 19 (20) |
36 40 49 (42) |
344 357 337 (346) |
45 68 60 (58) |
8 16 13 (12) |
|
1250.00 |
192 196 180 (189) |
28 25 15 (23) |
25 28 30 (28) |
342 328 331 (334) |
51 52 50 (51) |
13 7 12 (11) |
|
2500.00 |
171 182 189 (181) |
25 19 18 (21) |
44 43 32 (40) |
379 365 360 (368) |
57 48 51 (52) |
9 14 6 (10) |
|
5000.00 |
177 180 173 (177) |
27 13 17 (19) |
39 48 40 (42) |
336 376 354 (355) |
52 44 52 (49) |
16 7 6 (10) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
S9 Mix (-) |
Solvent control |
161 127 168 (152) |
24 17 24 (22) |
41 41 24 (35) |
222 255 206 (228) |
44 27 36 (36) |
18 13 8 (13) |
312.50 |
134 149 157 (147) |
25 19 27 (24) |
27 32 41 (33) |
128 194 218 (180) |
29 26 27 (27) |
8 19 16 (14) |
|
625.00 |
151 161 174 (162) |
27 25 24 (25) |
29 31 49 (36) |
293 351 314 (319) |
36 44 41 (40) |
12 15 17 (15) |
|
1250.00 |
137 140 162 (146) |
19 24 18 (20) |
26 25 37 (29) |
276 308 368 (317) |
44 33 38 (38) |
14 12 16 (14) |
|
2500.00 |
157 151 168 (159) |
17 19 20 (19) |
33 40 28 (34) |
234 256 236 (242) |
30 41 30 (34) |
17 15 13 (15) |
|
5000.00 |
149 159 164 (157) |
21 24 15 (20) |
30 30 36 (32) |
72 141 240 (151) |
32 21 38 (30) |
16 16 14 (15) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Positive control requiring S9 Mix |
Name |
2-Amino-anthracene |
Cyclophosphamide |
2-Amino-anthracene |
2-Amino-anthracene |
2-Amino-anthracene |
2-Amino-anthracene |
Concentration (µg/plate) |
2.50 |
400.00 |
50.00 |
20.00 |
2.50 |
2.50 |
|
Number of colonies/plate |
1284 916 1466 (1222) |
243 344 410 (332) |
737 847 894 (826) |
789 1028 1500 (1106) |
595 925 740 (753) |
139 167 147 (151) |
|
Positive control not requiring S9 Mix |
Name |
Sodium azide |
Sodium azide |
4-N00 |
Hitomycin-C |
2-Nitro-fluorene |
9-Amino-acridine |
Concentration (µg/plate) |
5.00 |
5.00 |
2.00 |
2.00 |
20.00 |
150.00 |
|
Number of colonies/plate |
834 831 676 (780) |
953 928 900 (927) |
552 620 488 (553) |
1497 1364 571 (1144) |
1805 1808 1686 (1766) |
1704 1357 1707 (1589) |
|
Notes: 1. When inhibition is found against growth of the bacteria, mark the applicable value with an asterix 2. Fill the average number of colonies in each concentration in the () 3. "Number of revertants"-Fi11 in the observed value and average value in order beginning with low concentration of the test substance |
|||||||
Toxicity test / Selection of concentrations
The selection of the highest scorable concentrations could not primarily be based on cytotoxicity data. Due to the presence of precipitates of the test substance on the microscopic slides, concentrations higher than 31.25 µg/mL (without metabolic activation) or 125 µg/mL (with metabolic activation) could not be scored. An inhibition in mitotic activity by 62.3% could be observed in the original experiment performed with metabolic activation (3h/15h) at the highest scorable concentration of 125 µg/mL. In the respective confirmatory experiment cytotoxicity was noted at the non-scorable concentration of 250 µg/mL only. In the absence of metabolic activation toxicity appeared only after 42 hours treatment at the highest concentration of 1000 µg/mL (non-scorable).
Original mutagenicity study
In the experiment performed without metabolic activation (experiment 1; 18 hours treatment), 2.0% of metaphases with specific chromosomal aberrations were detected in the negative control. At the concentrations of 7.81 µg/mL, 15.63 µg/mL and 31.25 µg/mL 1.0%, 2.0% and 1.5% of cells with specific chromosomal aberrations were found.
In the experiment performed with metabolic activation (experiment 2; 3 hours treatment/15 hours recovery), 1.0% of metaphases with specific chromosomal aberrations were seen in the negative control. At the concentrations of 31.25 µg/mL, 62.5 µg/mL and 125 µg/mL the respective values were 2.5%, 2.0% and 5.0%. The value obtained with the highest concentration of the second experiment showed a statistically significant difference when compared with the respective negative control. The value however is below the critical limit required for a positive response and it is within the historical range for negative controls. Furthermore no increase at all was observed in the respective confirmatory experiment. The slight increase in the frequency of aberrant metaphases is therefore considered to be spontaneous in origin.
Flow cytometry
The influence of the test substance on the cell cycle of CHO cells was tested at the concentrations selected for chromosome analysis. The DNA distribution was determined by flow cytometry and compared with the profile of the respective control culture. In the absence and in the presence of metabolic activation a shift in the DNA distribution profile could not be detected. Therefore, no evidence of a cell cycle disturbance by the test substance was obtained in the CHO cells.
Analytical results
The test material in suspension was analysed by UV/VIS-spectroscopy to confirm the intended concentrations to be used in the mutagenicity tests and the stability of the test substance in the vehicle used. The concentration values found were 120.6 and 124.1% of the calculated concentrations, thus indicating a sufficient stability of the test substance in the vehicle.
TABLE 1: MITOTIC INDEX VALUES / CYTOTOXICITY |
|||||
| Original study, experiment 1 18h treatment without metabolic activation | |||||
Cells scored |
Mitosis | M.I. % | Frequency % of control | ||
| Solvent control | 2000 | 263 | 13.15 | 100 |
|
| CGA65047SG100 (A-5787A) | |||||
| 1000 µg/mL | 2000 | 250 | 12.50 | 95.06 | |
| 500 µg/mL | 2000 | 234 | 11.70 | 88.97 | |
| 250 µg/mL | 2000 | 268 | 13.40 | 101.90 | |
| 125 µg/mL | 2000 | 273 | 13.65 | 103.80 | |
| 62.5 µg/mL | 2000 | 282 | 14.10 | 107.22 | |
| 31.25 µg/mL | 2000 | 290 | 14.50 | 110.27 | |
| 15.63 µg/mL | 2000 | 300 | 15.00 | 114.07 | |
| 7.81 µg/mL | 2000 | 310 | 15.50 | 117.87 | |
| Original study, experiment 2 3h treatment with metabolic activation/ 15h recovery | |||||
Cells scored |
Mitosis | M.I. % | Frequency % of control | ||
| Solvent control | 2000 | 220 | 11.00 | 100.00 | |
| CGA65047SG100 (A-5787A) | |||||
| 1000 µg/mL | 2000 | 114 | 5.70 | 51.82 | |
| 500 µg/mL | 2000 | 70 | 3.50 | 31.82 | |
| 250 µg/mL | 2000 | 62 | 3.10 | 28.18 | |
| 125 µg/mL | 2000 | 83 | 4.15 | 37.73 | |
| 62.5 µg/mL | 2000 | 218 | 10.90 | 99.09 | |
| 31.25 µg/mL | 2000 | 224 | 11.20 | 101.82 | |
| 15.63 µg/mL | 2000 | 238 | 11.90 | 108.18 | |
| 7.81 µg/mL | a) | ||||
a) When three subsequent concentrations with a frequency of 70% mitosis or more in relation to the solvent control are found, the evaluation of the lower concentrations is omitted.
| TABLE 2 ORIGINAL MUTAGENICITY STUDY, EXPERIMENT 1 | ||||||||||
| 18 h treatment without metabolic activation | ||||||||||
| Treatment | total no of cells examined | % cells with specific aberrations # | total number of cells with aberrations | |||||||
| gaps | ct del | ct exc | cs del | cs exc | mab | pol | end | |||
| Solvent control | 200 | 2.0 | 1 | 2 | 1 | 1 | 7 | |||
| CGA 65047 SG 100 (CA-5787 A) | ||||||||||
| 7.81 µg/mL | 200 | 1.0 | 3 | 1 | 1 | 3 | ||||
| 15.63 µg/mL | 200 | 2.0 | 2 | 1 | 2 | 1 | 3 | |||
| 31.25 µg/mL | 200 | 1.5 | 3 | 2 | 1 | 6 | ||||
| positive control (Mito-C, 0.2 µg/mL | 50 ) | 24.0*** | 5 | 6 | 6 | 2 | ||||
| TABLE 3 ORIGINAL MUTAGENICITY STUDY, EXPERIMENT 2 | ||||||||||
| 3 h treatment with metabolic activation / 15 h recovery | ||||||||||
| Treatment | total no of cells examined | % cells with specific aberrations # | total number of cells with aberrations | |||||||
| gaps | ct del | ct exc | cs del | cs exc | mab | pol | end | |||
| Solvent control | 200 | 1.0 | 6 | 2 | 8 | |||||
| CGA 65047 SG 100 (CA-5787 A} | ||||||||||
| 31.25 µg/mL | 200 | 2.5 | 6 | 2 | 2 | 1 | 3 | 1 | ||
| 62.5 µg/mL | 200 | 2.0 | 13 | 1 | 3 | 9 | ||||
| 125 µg/mL | 200 | 5.0* | 10 | 6 | 3 | 1 | 5 | 1 | ||
| positive control (CPA, 20 µg/mL) | 100 | 17.0*** | 2 | 6 | 9 | 3 | 3 | |||
Legend to Tables2 -3 |
|
| ctdel | Chromatid deletions (including deletions, breaks, fragments) |
| ct exc | Chromatid exchanges (including triradials, quadriradials, endfusions and acentric rings) |
| csdel | Chromosome deletions (including deletions, breaks, fragments) |
| cs exc | Chromosome exchanges (including dicentrics, polycentrics, centric and acentric rings) |
| mab | Multiple aberrations: metaphases containing more than10 aberrations of different types or more than 5 aberrations of one particular type (excluding gaps) |
| gaps | Chromatid and chromosome type gaps |
| pol | Polyploid metaphases (>30 centromers) |
| end | Endoreduplications |
| CPA | Cyclophosphamide |
| Mito-C | Mitomycin-C |
| *) | Statistical significance:0.05 =P> 0.01 |
| Statistical significance:0.01 =P> 0.001 | |
| Statistical significance: P< 0.001 | |
| #) | %cells with aberrations excluding gaps and numerical alterations(pol,end) |
Toxicity
In the cytoxicity range-finder experiment, 12 concentrations of the test substance were tested. In the part with metabolic activation, the concentrations ranged from 0.49 to 1000.0 µg/mL separated by 2 fold intervals. 1000.0 µg/mL represents the highest applicable concentration to be tested in the experiment. In the part with metabolic activation, down to the concentration of 31.25 µg/mL, a growth inhibiting effect greater than 50.0% in comparison to the negative control could be seen. No relevant cytotoxicity could be detected after treatment with the test chemical in the part without metabolic activation.
Accordingly, five concentrations were selected for the original mutagenicity experiment. In the part with metabolic activation the following concentrations were selected: 0.49, 1.95, 7.81, 31.25 and 125.0 µg/mL. At the top concentration of 125.0 µg/mL the mean zero hour survival value was 49.33%. The relative total growth at the end ofthe expression period revealed a mean value of 31.40%. In the part without metabolic activation the concentrations appplied were: 3.91, 15.63, 62.50, 250.0 and 1000.0 µg/mL. 1000.0 µg/mL represents the highest applicable concentration to be tested in the experiment. The highest concentration showed a mean relative survival of 62.01%. The mean relative total growth value was 80.31%. All concentrations were selected to determine viability and TFT-resistance two days after treatment.
In the confirmatory mutagenicity experiment, in the part with metabolic activation, the concentration range was increased. The concentrations applied were: 0.98, 3.91, 15.63, 62.50 and 250.0 µg/mL. In the part without metabolic activation the same concentration range as in the original experiment was selected. The mean relative survival value obtained in the part with metabolic activation was 47.45% at the highest concentration. The mean relative total growth value obtained after the two days expression period was 42.05%. The zero hour survival value in the part without metabolic activation was 81.90%. The relative total growth determined after the expression revealed a value of 75.74%. All concentrations were selected to determine viability and TFT-resistance 2 days after treatment.
Mutagenicity
In the presence and absence of metabolic activation, in the original and confirmatory experiments, reproducible, statistically significant and dose-related relevant increases in mutant frequencies were not observed. In the original experiment, in the part with metabolic activation, the highest concentration of 125.0 µg/mL was excluded from statistical analysis due excessive heterogeneity in the duplicate survival plates. In the part without metabolic activation the concentration of 15.36 µg/mL was excluded from statistical anlysis due to heterogeneity in the survival plates. Nevertheless, the mutant frequency values found at these concentrations are lying within the normal range and do not influence the validity of the study in any respect.
Large and small colonies
For the negative and positive controls the number of wells containing small colonies and the number of wells containing large colonies were scored. With metabolic activation the mean proportion of small colonies in the negative controls ranged from 40.0 to 44.4%, whereas with the positive controls (DMN) an increased proportion of 55.4 to 59.5% was observed. Without metabolic activation the proportion of small colonies in the negative controls ranged from 67.3 to78.0%, whereas with the positive controls (EMS) a proportion of 42.0 to 53.3% was observed.
Small and large colony numbers are not reported for CGA 65 047 SG 100, (A-5787 A) as the data did not fulfil the criteria for a positive response.
Table 1: Summary of the mutagenicity test (Experiment with metabolic activation)
| Treatment | Zero hour survival % | Relative total growth (RTG) % |
| Negative control DMN 2 µL/mL | 100.00 78.35 |
100.00 48.88 |
| CGA 65 047 SG 100 (A-5787 A): | ||
| 125.00 µg/mL 31.25 µg/mL 7.81 µg/mL 1.95 µg/mL 0.49 µg/mL |
49.33 103.76 93.79 85.79 75.22 |
31.40 86.83 64.80 63.13 60.61 |
| Treatment | Mutant frequency X 10E-6 | Significance (P) |
| Negative control DMN 2 µL/mL | 24.58 387.77 |
# |
| CGA 65 047 SG 100. (A-5787 A): | ||
| 125.00 µg/mL 31.25 µg/mL 7.81 µg/mL 1.95 µg/mL 0.49 µg/mL |
54.08 27.73 25.17 36.01 27.83 |
§ NS NS NS NS |
| Test for linear trend: NS | ||
Table 2: Summary of the mutagenicity test (Experiment without metabolic activation)
| Treatment | Zero hour survival % | Relative total growth (RTG) % |
| Negative control EMS 1 µL/mL | 100.00 1.82 |
100.00 2.54 |
| CGA 65 047 SG 100 (A-5787 A): | ||
| 1000.00 µg/mL 250.00 µg/mL 62.50 µg/mL 15.63 µg/mL 3.91 µg/mL |
62.01 74.51 100.13 134.44 144.04 |
80.31 109.86 96.75 92.87 115.83 |
| Treatment | Mutant frequency x 10E-6 | Significance (P) |
| Negative control EMS 1 µL/mL | 33.76 841.91 |
# |
| CGA 65 047 SG 100 (A-5787 A): | ||
| 1000.00 250.00 µg/mL 62.50 µg/mL 15.63 µg/mL 3.91 µg/mL |
38.61 22.40 35.37 40.38 39.89 |
NS NS NS § NS |
| Test for linear trend | NS |
§ excluded from experiment;
NS not significant.
Dose-dependent positive responses to zinc acetate were obtained in the presence and absence of the S9 activation system, although the S9 reduced both the clastogenic response and the toxicity.
Severe aberrations such as dicentric chromosomes were recorded only in lymphocyte cultures treated with the lowest concentration of zinc chloride (3 x 10E-5 M) added at time 0, regardless whether the cultures were fixed after 48 or 72 h.
The most common aberration found for all tested metal salts was the occurrence of chromosome fragments. Dicentric chromosomes were only recorded in lymphocyte cultures treated with the lowest concentration of zinc chloride (3x10E-5 M) added at time 0, regardless whether the cultures were fixed after 48 or 72 h.
The chi-square analysis did not show that the incidence of dicentrics in the cells treated with zinc were significant when tested against the controls only. If, however, controls combined with the cadmium and lead exposed were tested against all zinc treated lymphocytes the difference was highly significant (P = 0.004).
Table 1.Results of Mutation Test with zinc sulphate
Dose (mg/plate) |
No of Revertants /plate |
|||
Zinc sulphate |
||||
|
TA97 |
TA102 |
||
|
-S9 |
+S9 |
-S9 |
+S9 |
10 |
117 |
136 |
185 |
412 |
5 |
115 |
169 |
243 |
481 |
1 |
125 |
158 |
323 |
435 |
0.5 |
132 |
155 |
319 |
452 |
0.1 |
143 |
176 |
295 |
437 |
0 |
134 |
185 |
318 |
465 |
Positive control |
229 |
3,118 |
4,162 |
2,646 |
Solvent: DW (not specified) |
||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Cytogenicity (mouse)
ZnCl2, chromosomal aberration, negative (Deknudt and Gerber, 1979)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to Read Across Statement attached in Section 13
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Remarks on result:
- other: in mice with normal diet
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Remarks on result:
- other: in mice with calcium deficient diet
- Additional information on results:
- Treatment with heavy metals as well as the low-calcium diet significantly reduced the body weight of the mice, and the effect was more pronounced when treatments with heavy metals and low calcium diet were combined. In general, the intoxicated animals on a low calcium diet had lost weight, were weak, seemed anaemic and had brittle femurs. Serum calcium was reduced in animals on a low-calcium diet, and this effect was accentuated by intoxication with heavy metals whereas this intoxication as such did not significantly influence calcium levels in animals on a normal diet. The number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.
The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc. - Conclusions:
- The present experiments confirm that zinc can cause severe chromosomal anomalies in animals kept on a low calcium diet. The number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.
- Executive summary:
Mice kept on a normal (1.1% calcium) or low-calcium (0.03%) diet were exposed for one month to zinc chloride (0.5% Zn). The concentrations, given in a poor calcium diet, represent a LD 50 (30 days). After the mice were killed bone-marrow cells were assayed for chromosomal aberrations, and serum calcium was determined. The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc. The number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
Reference
Treatment and diet |
Body weight (g) |
Serum calcium (mg/100ml) |
Cells with structural aberrations |
Type and ChromatidGaps |
||||
Chromatid aberrations |
Chromatid aberrations |
|||||||
gaps |
Breaks |
Gaps |
Fragments |
Dicentrics |
||||
Control+ Ca |
29.90±0.12 |
10.24±0.06 |
1.80±0.60 |
1.20±0.49 |
|
|
0.6±0.35 |
|
Control - Ca |
21.80±0.27 !! |
9.45±0.15 !! |
2.00±0.63 |
1.80±0.60 |
|
|
0.4±0.28 |
|
Zinc+ Ca |
17.90±0.23 ** |
9.76±0.29 *! |
2.80±0.75 |
1.80±0.60 |
0.2±0.2 |
|
0.4±0.28 |
0.4±0.28 |
Zinc - Ca |
12.05±0.25 **!! |
8.76±0.24 |
5.00±1.00 ** |
3.20±0.80 |
|
0.4±0.28 |
0.6±0.35 |
1.2±0.49 |
a All values represent means -+ standard errors (Poisson errors for counting data). Statistically significant differences from the respective controls without heavy metals are indicated as * and ** for the p <= 0.05 and p <= 0.01 levels. Differences from the respective treated group with calcium in the diet are shown as ! and !! for the p <= 0.05 and p <= 0.01 levels. b Exact probability 0.06 |
||||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Salmonella and E. coli tests in vitro (Ames Tests):
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to induce mutagenic effect in histidine-requiring strains of Salmonella typhimurium (TA 98, TA 100, TA 102, TA 1535, TA 1537) and in a tryptophan-requiring strain of Escherichia coli (WP2 uvr A) (OECD Guideline 471) (CIBA-GEIGY, 1994b; Report No. 931146a). The test compound was dissolved in bidistilled water and tested at five concentrations ranging from 312.5 to 5000 µg/plate with and without metabolic activation. Suitable positive controls were used for each strain. All experiments were repeated in order to confirm the results. The results revealed no increased incidence of mutants by the test item with and without metabolic activation. Therefore, it was concluded that the test compound did not show mutagenic activity in S. typhimurium and E. coli. The positive controls induced mutagenic activity. In conclusion, Fe(Na)EDDHA provoked no mutagenic activity in this test system.
Zinc sulphate (Fujita 1988)
Mutagenicity zinc sulphate was examined in Ames' tester strains,Salmonella typhimurium TA97 and TA102. The mutation test was carried out by the preincubation procedure described by Ames et al. The test chemicals were preincubated with S9 mix or phosphate buffer (pH7.4) for 20 min. Zinc sulphate was negative in two tester strains.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
Zinc acetate (Thompson 1989)
Ames test was performed with zinc acetate. The plate-incorporation assay was performed with Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The results of the Salmonella/mammalian microsome plate-incorporation assay for zinc acetate were uniformly negative. Zinc acetate was neither toxic (determined by reduction of bacterial lawn) nor mutagenic to any of the 5 strains tested over a dose range of 50-7200 µg/plate.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
Mammalian Chromosome Aberation Test
1. In-vitro
EDDHA
The test compound Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to provoke clastogenic effects in Chinese hamster ovary cells (CCL61) in vitro (OECD TG 473) (CIBA-GEIGY, 1994c; Report No. 931147). The compound was dissolved in DMSO and tested without metabolic activation at concentrations of 0, 7.81,15.63 and 31.25 µg/mL for 18 and 42 hours. With metabolic activation (liver S9 fraction from Aroclor 1254 induced rat liver) concentrations of 0, 31.25, 62.5 and 125 µg/mL were applied for 3 hours followed by 15 hours recovery or 3 hours followed by 39 hours recovery. Higher concentrations could not be reached due to solubility limitations. Three independent experiments of each with and without metabolic activation were performed. Two replicate culture per concentration and 200 cells per concentration were evaluated.
The results showed in both experiments with and without metabolic activation no increased number of metaphases with chromosomal aberrations. In contrast, the positive controls (Mitomycin 0.2 µg/mL and Cyclophosphamide 20 µg/mL) induced clastogenic effects. In conclusion, the test substance provoked no clastogenic activity in this test in vitro.
Zinc chloride (Deknudt and Deminatti 1978)
Chromosome aberrations in human lymphocytes induced by zinc chloride were analysed. The results suggest that high levels of zinc (3 x 10E-3 M) are cytotoxic and that lower concentration (3 x 10E-5) can cause severe chromosome aberrations (dicentrics). It must be pointed out, however, that the concentration of zinc used in the present experiments are extremely high representing up to 1000 times the respective concentrations reported in the blood of people professionally contaminated by heavy metals and which have been shown to be 22.4 µg% for zinc.
Due to the fact that the Zn ion is a constituent of the target substance, the result is relevant for the target substance.
However, in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions. This is confirmed by a negative in vivo micronucleus assay with zinc chloride (Deknudt and Gerber, 1979, see below).
Zinc acetate (Thompson 1989)
A chromosome aberration assay with zinc acetate was conducted in Chinese Hamster Ovary cells. Zinc acetate induced chromosome aberrations in the presence and absence of metabolic activation. These results indicate that zinc is an effective clastogen when presented to a susceptible cell population in an appropriate form.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
However, in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions. This is confirmed by a negative in vivo micronucleus assay with zinc chloride (Deknudt and Gerber, 1979, see below).
2. in vivo
Zinc chloride (Deknudt and Gerber 1979)
Mice kept on a normal (1.1% calcium) or low-calcium (0.03%) diet were exposed for one month to zinc chloride (0.5% Zn). The concentrations, given in a poor calcium diet, represent a LD 50 (30 days). After the mice were killed bone-marrow cells were assayed for chromosomal aberrations, and serum calcium was determined. The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc. The number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
In-vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay):
EDDHA
Fe(Na)EDDHA (CAS 84539 -55 -9) was tested for the ability to provoke mutations at the tk locus in L5178Y mouse lymphoma cells in vitro (OECD Guideline 476) (CIBA-GEIGY, 1994a; Report No. 931146b). The test compound was dissolved in DMSO. The range finding experiments showed that 1000 µg/mL was the highest concentration which could be used. Higher concentrations (greater than 100 mg/mL) produced precipitates in the vehicle. In the presence of metabolic activation (liver S9 -fraction from Aroclor 1254 treated rats) the two highest concentrations revealed cytotoxicity. In absence of metabolic activation no toxicity was noted.
For the mutagenicity experiment, concentrations ranging from 0 to 125 µg/mL with metabolic activation and from 0 to 1000 µg/mL without metabolic activation were used. In the confirmatory experiment with metabolic activation concentrations ranging from 0 to 250 µg/mL were applied. The same concentrations (0 to 1000 µg/mL) were used in the confirmatory experiment without metabolic activation. Corresponding positive controls (N-Nitrosodimethylamine, with metabolic activation and Ethylmethansulfonate without metabolic activation) were included. The mouse lymphoma cells were treated for 4 hours. After two days expression time, mutations at the tk locus were selected by resistance to 5-trifluorothymidine. Two types of colonies were selected, large colonies (base-pair substitutions and deletions) and small colonies (chromosome aberrations). The results showed no increased incidence of mutations at the tk locus of mouse lymphoma L5178Y cells in presence or absence of metabolic activation. Positive controls showed mutagenic activity. In conclusion, Fe(Na)EDDHA was not mutagenic in this test system in vitro.
Zinc chloride (Amacher and Paillet 1980)
Zinc chloride was tested for the potential to induce trifluorothymidine-resistant (TFT Res) mutants in L5178Y/TK+/- mouse lymphoma cell by directly exposing cells to varied doses for 3 h. 48 h after treatment, metal-treated cells and solvent controls were cloned in soft-agar media and plated. Trifluorothymidine resistance (TFT Res) was determined by adding 4 µg/ml TFT to one set of plates. All colonies growing either in the presence of TFT (TFT Res) or its absence (viable count colonies) were counted on day 7 after incubation at 37°C. Those TFT Res colonies which were equivalent in size to colonies growing in the solvent control viable count plates i.e., large, were scored as mutants. The result was negative for ZnCl2.
Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance.
Zinc acetate (Thompson 1989)
A mouse lymphoma TK+/- assay was perormed with zinc acetate. The results indicate zinc is an effective mutagen when presented to a mouse lymphoma cells in an appropriate form. Due to the fact that the Zn ion is a constituent of the target substance, the result is also relevant for the target substance. However, controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.
Conclusion
While the EDDHA ligand is clearly negative in in vitro genetic toxicity tests, the in vitro tests of zinc salts had conflicting results. Postive indication for genotoxicity in mammalian cells and cytogenicity were found in in vitro studies. However, these results were not confirmed by in vivo studies. No chromosomal aberrations were found in vivo in mice under normal feeding conditions (Deknudt and Gerber, 1979). This shows that genotoxicity of zinc does only occur at overload conditions. This may lead to a cellular stress response and to the generation of reactive oxygen species. Therefore, the generation of reactive oxygen species may the primary mechanism of action of zinc with regard to genotoxicity. As mammals have effective mechanisms to regulate zinc homeostasis, they are able to prevent toxic overload conditions. This is confirmed by the absence of chromosomal aberrations in the available in vivo study under normal feeding conditions (Deknudt and Gerber, 1979). In mammals zinc is a co-factor in over 200 enzyme systems and is involved in stabilizing DNA by binding to the phosphate portions. The protective effect of zinc against cadmium-induced DNA strand breaks on has also been shown by Coogan et al. (1992). Thus zinc is an essential element for cells, stabilizing the DNA. With regard to the target substance, it has to be considered that it is a dimeric complex with a high molecular weight and a low logPow. Therefore, in its complexed form, it is unlikely to pass the cell membrane. Assuming a dissociation of the complex, an extremely high amount of the target substance would be required to release a toxic concentration of elemental zinc.
Justification for classification or non-classification
Based on these results the substance is not classified according to Regulation (EC) No 1272/2008.
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