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EC number: 231-203-4 | CAS number: 7446-26-6
- Life Cycle description
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- Short-term toxicity to fish
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Endpoint summary
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Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
bacterial gene mutation (OECD 471, RL1): not mutagenic
mammalian gene mutation (OECD 490, RL1): not mutagenic
cytogenicity study (OECD 487 (MNT), RL1): positive without metabolic activation; negative with metabolic activation
Read across data with zinc chloride and zinc sulfate showed negative and ambigous results in chromosome aberration assays.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 Jan 2016-18 Feb 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Adopted 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- First experiment (plate incorporation method): 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg/plate with and without metabolic activation
Second experiment (preincubation method): 31.6, 100, 316, 1000, 3160 and 5000 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 0.05 M HCl
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the solvents recommended: water, dimethylsulfoxide (DMSO), ethanol or acetone, but was completely soluble at 3.16 mg/mL 0.05 M HCl in preliminary solution tests. For concentrations lower than or equal to 316 μg/plate, the test item was completely dissolved. For concentrations of 1000, 3160 and 5000 μg/plate the test material was suspended in 0.05 M HCl solution. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.05 M HCl
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-amino-anthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: 2 replications each in 2 independent experiements
DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn. Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn. - Evaluation criteria:
- A test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537, and 1.5-fold of the solvent control for TA102 in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) is observed.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first. A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test. - Statistics:
- Mean values and standard deviation were calculated.
- Key result
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at the top concentration of 5000 μg/plate in all test strains
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test material precipitation was noted in all experiments at concentrations of 3160 and 5000 μg/plate.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
Negative Reference Item
Strain TA 98 TA 100 TA 102 TA 1535 TA 1537
S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 29.9 31.3 148.5 149.2 275.5 279.8 19.4 19.2 6.4 6.5
SD 5.7 5.9 18.1 18.2 15.5 16.2 4.4 4.4 1.8 1.9
Min 19 14 103 106 240 245 10 8 2 0
Max 49 49 191 195 319 319 34 42 10 10
Positive Reference Item
Strain TA 98 TA 100 TA 102 TA 1535 TA 1537
S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
2-nitro- Benzo[a] Sodium 2-amino- Mitomycin Benzo[a] Sodium 2-amino- 9-amino- Benzo[a]
fluorene pyrene azide anthracene C pyrene azide anthracene acridine pyrene
Mean 148.4 147.3 928.7 937.9 1014.6 1008.9 133.2 133.3 81.1 81.5
SD 33.6 33.7 107.8 99.8 121.4 109.5 28.9 29.5 28.8 28.9
Min 91 91 463 703 756 757 51 49 26 28
Max 305 315 1209 1181 1637 1571 220 271 178 189 - Conclusions:
- Interpretation of results: the test material dizinc pyrophosphate is negative for mutagenicity (non-mutagenic) with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 Jan 2016-25 Apr 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- Adopted 29 Jul 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: ATCC, 0801 University Blvd., Manassas, VA, USA
- Suitability of cells: mouse lymphoma L5178Y cells heterozygous at the TK locus (+/-)
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Growth medium: RPMI 1640 with glutamax supplemented with pluronic F68, gentamycin, amphotericin B, and horse serum (10% by volume). Treatment medium: growth medium with horse serum (5% by volume). Plating medium: growth medium without pluronic F68 and increased horse serum. Selection medium: growth medium containing 3 µg/mL of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- Concentrations were selected using the following criteria: At least four analysable concentrations based on results of the preliminary cytotoxicity study, appropriately spaced (separation factor of √10) in the relative growth range of ≥10-100%.
Doses for the mutagenicity study were:
3 h exposure without and with metabolic activation: 0.316, 1.0, 3.16, 10.0 and 31.6 µg/mL medium
24 h exposure without metabolic activation: 0.1, 0.316, 1.0, 3.16 and 10.0 µg/mL medium - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 0.05 M HCl
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the solvents recommended: water, dimethylsulfoxide (DMSO), ethanol or acetone, but was completely soluble at 2 mg/mL 0.05 M HCl solution. This suspension then was further diluted with culture medium to obtain the final concentration desired in the exposure medium. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.05 M HCl
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: first experiment: 3 h exposure with and without S9, and second experiment: 3 h and 24 h exposure without S9
- Expression time (cells in growth medium): 2 days after the the end of treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well plates containing 3 µg/mL TFT selection medium. These plates were incubated for 11-14 days.
- Selection time (if incubation with a selection agent): 11-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-16 days
SELECTION AGENT (mutation assays): 5-trifluoro-thymidine (TFT) 3 µg/mL medium
NUMBER OF REPLICATIONS: duplicates in 96-well plates during survival and expression periods, then quadruplicates in 96-well plates for the selection period
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: cloning efficiency and relative total growth
- OTHER:
Small and large colonies were differentiated, as small colonies are indicative of clastogenic effects such as chromosomal abberations. - Evaluation criteria:
- The test material is considered to be clearly positive if, in any of the experimental conditions examined, the increase in mutation frequency is above the concurrent control (i.e.; historic control), and the increase is concentration related (i.e.; dose dependent trending).
- Statistics:
- Means for the individual control data, percent calculations for survival and plating efficiencies, ratios of large versus small colonies.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 3 h exp: >10 µg/mL and at 24 h exp: >3.16 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity study, with and without metabolic activation, was conducted to establish the top concentration for the main study. Based on the results of the repeated preliminary cytotoxicity study, top concentrations of 31.6 μg dizinc pyrophosphate/ mL medium for the 3-hour exposures with and without metabolic activation, and 10.0 μg/mL for the 24-hour exposure without metabolic activation were used in the mutagenicity tests. Cytotoxicity was noted in the 3-hour exposures without and with metabolic activation at the top concentration of 31.6 μg test material/mL medium, and in the 24-hour exposure at the top concentration of 10.0 μg test material/ mL medium.
HISTORICAL CONTROL DATA (Means and standard deviation (with ranges))
- Positive historical control data: Number of Mutant Colonies per 1E+06 cells: (Mean value ± SD (with ranges))
-- 3-hour exposures (-S9) Methylmethanesulfonate: 1523.8 ± 667.9 (436.1-2712.5)
-- 3-hour exposures (+S9) 3-Methylcolanthrene: 1344.5 ± 642.7 (415.7-3466.7)
-- 24-hour exposures (-S9) Methylmethanesulfonate: 1647.1 ± 709.7 (555.7--2939.4).
- Negative (solvent/vehicle) historical control data: Number of Mutant Colonies per 1E+06 cells: (Mean value ± SD (with ranges))
-- 3-hour exposures (-S9): Negative control: 86.4 ± 32.6 (50.2-168.2); Negative control Aqueous (DI H2O/ 0.05 M HCl) Solvent: 95.4 ± 42.3 (54.6-160.5); Negative control Organic (DMSO) Solvent: 86.6 ± 31.2 (50.2-160.5)
-- 3-hour exposures (+S9): Negative control: 89.3 ± 33.7 (50.0-169.5); Negative control Aqueous (DI H2O/ 0.05 M HCl) Solvent: 101.9 ± 40.6 (50.4-169.5); Negative control Organic (DMSO) Solvent: 74.8 ± 15.7 (51.8-113.0)
-- 24-hour exposures (-S9): Negative control: 87.4 ± 32.7 (50.9-161.5); Negative control Aqueous (DI H2O/ 0.05 M HCl) Solvent: 89.4 ± 41.7 (51.0-161.5); Negative control Organic (DMSO) Solvent: 94.3 ± 30.9 (62.0-161.5) - Conclusions:
- Interpretation of results: the test material dizinc pyrophosphate, tested up to cytotoxic concentrations in two independent experiments, with and without metabolic activation, was negative with respect to inducing mutations or chromosomal abberations (no change was noted in the ratio of small to large mutant colonies) in the L5178Y TK +/- mammalian cell mutagenicity test.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 25 Jan 2016-08 Apr 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- Adopted 23 Jul 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: human peripheral
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: young human donors
- Suitability of cells: peripheral blood collected by venipuncture
- Sex, age and number of blood donors if applicable: male or female, 18-35 years of age, healthy, non-smoking
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Initially innocula were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin, sealed and incubated at 37°C. During exposure fresh Ham's F10 medium with fetal calf serum without and with S9 was used. During mitotic arrest the medium was replaced with Chromosome medium containing 5 µg/mL Cytochalasin B.
- Properly maintained: yes - Cytokinesis block (if used):
- 5 µg/mL Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of male rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- Concentrations were selected using the following criteria: At least three analysable concentrations based on results of the preliminary cytotoxicity study, appropriately spaced (separation factor of √10), and the highest concentration should aim to produce 55 ± 5% cytotoxicity. Since cytotoxicity was observed at 100 µg/mL, the next highest level was chosen as the top concentration.
4 h treatment (with and without metabolic activation): 6.25, 12.5, 25 and 50 µg/mL
24 h treatment (without metabolic activation): 6.25, 12.5, 25 and 50 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 0.05 M HCl
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the solvents recommended: water, dimethylsulfoxide (DMSO), ethanol or acetone, but was completely soluble at 3.16 mg/mL 0.05 M HCl in preliminary solution tests. This suspension then was further diluted with culture medium to obtain the final concentration desired in the exposure medium. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.05 M HCl
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: colchicine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4, and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 25 h; 24 h treatment: 45 h.
SPINDLE INHIBITOR (cytogenetic assays): CytoB 5 µg/mL
STAIN (for cytogenetic assays): 10% Giemsa
NUMBER OF REPLICATIONS: 2 cultures per concentration (main study), one culture per concentration (preliminary test)
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Following final fixation, two drops per culture, air-dried on clean slide at room temperature, stained with 10% Giemsa.
NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration)
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction.
DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index (CBPI) to calculate cell proliferation. Replicative Index (RI) to calculate the % cytostasis. - Evaluation criteria:
- A test material is considered to be clearly positive if: a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control and b) the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test and c) any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits).
A test material is considered clearly negative if: a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control and b) there is no concentration-related increase when evaluated with an appropriate trend test and c) all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits). - Statistics:
- Chi-square test corrected for continuity.
Regression analysis by Spearman's rank correlation coefficient (p ≤ 0.05). - Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- 24 h and repeated 24 h
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 50 µg/mL: highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- 4 h
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 50 µg/mL: highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: The concentrations employed were chosen based on the results of a preliminary cytotoxicity study where complete cytotoxicity was noted at concentrations ≥100 μg dizinc pyrophosphate/ mL medium. In the main study, 50 μg/mL was employed as the top concentration for the genotoxicity tests without and with metabolic activation, and 35 μg/mL were employed as the top concentration for the repeat genotoxicity test without metabolic activation (24-hour exposure). In the main study, cytotoxicity was noted in the experiments without and with metabolic activation at the top concentrations of 35 or 50 μg test material/ mL medium.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Micronucleus frequency per 1000 cells -- (-S9): Mitomycin C: 46.9 ± 35.0 (17 - 137); Colchicine: 25.9 ± 9.5 (15-63); (+S9): Cyclophosphamide: 44.0 ± 37.7 (14-158)
- Negative (solvent/vehicle) historical control data: Micronucleus frequency per 1000 cells -- (-S9): Untreated control: 6.6 ± 2.9 (2-17 with 95% CI 5.9-7.3); Vehicle control: 6.3 ± 3.1 (2-18 with 95% CI 5.7-6.8); (+S9): Untreated control: 6.4 ± 2.6 (3-13 with 95% CI 5.7-7.1); Vehicle control: 6.1 ± 4.2 (1-22 with 95% CI 5.1-6.7) - Conclusions:
- Under the conditions of this test, dizinc pyrophosphate, tested up to cytotoxic concentrations in the absence and presence of metabolic activation, at an exposure time of 4 hours, revealed no indications of chromosomal damage in the in vitro micronucleus test.
Dizinc pyrophosphate tested up to cytotoxic concentrations in the absence of metabolic activation, at an exposure time of 24 hours, revealed a clearly positive, and concentration dependent response.
Hence, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system under these test conditions. - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- Summary of available data used for the endpoint assessment of the target substance
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- refer to analogue justification in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Evaluation criteria:
- not specified
- Key result
- Species / strain:
- other: human dental pulp cells (D824)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: Someya, 2008, zinc chloride
- Conclusions:
- Interpretation of results: negative
- Executive summary:
As explained in the analogue justification, zinc is considered to be the toxic element. Therefore, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity potential.
Referenceopen allclose all
Dizinc pyrophosphate
EXPERIMENT 1(Standard Plate Test, SPT) |
|||||
S9-Mix |
Without |
||||
Test Item (mg/plate) |
Base-pair substitution type |
Frameshift type |
|||
TA 100 |
TA 102 |
TA 1535 |
TA 98 |
TA 1537 |
|
VC |
135.0±9.2 |
268.3±5.5 |
17.3±1.5 |
31.7±6.1 |
5.7±1.5 |
31.6 |
149.3±3.5 |
264.7±3.8 |
14.3±0.6 |
31.3±8.3 |
4.0±0.0 |
100 |
167.7±9.0 |
263.0±1.0 |
16.3±1.5 |
29.7±4.9 |
7.0±1.7 |
316 |
150.7±20.8 |
267.0±1.0 |
16.7±1.5 |
32.7±4.9 |
4.7±1.5 |
1000 |
126.3±7.6 |
276.7±9.5 |
15.0±1.0 |
32.7±1.5 |
6.7±3.5 |
3160 |
164.7±13.3 |
268.0±7.8 |
15.0±1.0 |
30.7±7.1 |
5.0±1.0 |
5000 |
17.3±4.0 |
21.7±1.5 |
6.3±1.5 |
9.0±1.0 |
1.3±0.6 |
PC (mg/plate) |
NaN3(10) |
MMC (10) |
NaN3(10) |
2NF (10) |
AAC (100) |
916.3±10.7 |
1018.3±9.5 |
143.3±1.5 |
177.0±3.0 |
51.70±3.1 |
|
S9-Mix |
With |
||||
Test Item (mg/plate) |
Base-pair substitution type |
Frameshift type |
|||
TA100 |
TA 102 |
TA 1535 |
TA98 |
TA1537 |
|
VC |
149.7±3.8 |
251.7±1.5 |
22.0±0.0 |
36.0±2.6 |
6.0±1.0 |
31.6 |
166.3±10.2 |
259.0±0.0 |
18.0±0.0 |
29.0±2.0 |
6.7±3.1 |
100 |
145.7±9.0 |
270.7±9.2 |
17.3±1.5 |
32.0±3.6 |
5.3±1.5 |
316 |
142.3±8.1 |
297.0±19.1 |
14.7±0.6 |
25.7±3.8 |
6.0±1.0 |
1000 |
134.7±7.6 |
253.7±5.5 |
16.0±2.6 |
27.3±6.0 |
5.7±1.5 |
3160 |
150.0±14.0 |
256.3±3.2 |
16.0±2.6 |
24.7±3.1 |
6.0±1.0 |
5000 |
27.3±5.7 |
24.7±6.4 |
5.7±1.2 |
8.0±1.0 |
1.0±0.0 |
PC (mg/plate) |
AAN (2) |
B[a]P (10) |
AAN (2) |
B[a]P (10) |
B[a]P (10) |
878.0±37.6 |
1022.3±26.3 |
141.7±1.2 |
177.0±2.0 |
52.7±3.1 |
VC = Vehicle control; PC = Positive control
NaN3= Sodium azide
2NF = 2-Nitrofluorene
MMC = Mitomycin C
AAC = 9-Aminoacridine
AAN = 2-Aminoanthracene
B[a]P = Benzo[a]pyrene
EXPERIMENT 2(Standard Plate Test with Incubation) |
|||||
S9-Mix |
Without |
||||
Test Item (mg/plate) |
Base-pair substitution type |
Frameshift type |
|||
TA100 |
TA 102 |
TA 1535 |
TA98 |
TA1537 |
|
VC |
145.0±20.0 |
281.7±23.5 |
21.7±8.1 |
23.7±2.1 |
8.3±0.6 |
31.6 |
155.7±4.9 |
254.7±1.2 |
20.0±1.7 |
28.3±1.5 |
6.3±0.6 |
100 |
141.0±21.1 |
254.3±2.1 |
18.3±0.6 |
27.0±3.5 |
5.7±1.5 |
316 |
138.3±15.5 |
282.3±22.8 |
16.0±1.7 |
27.7±3.2 |
8.3±1.2 |
1000 |
151.7±4.9 |
260.7±15.6 |
15.0±1.0 |
25.3±3.2 |
6.0±1.7 |
3160 |
135.7±12.4 |
268.0±2.6 |
22.3±5.9 |
26.3±1.2 |
6.3±0.6 |
5000 |
32.0±10.5 |
104.70±3.5 |
6.3±1.2 |
8.3±1.5 |
1.3±0.6 |
PC (mg/plate) |
NaN3(10) |
MMC (10) |
NaN3(10) |
2NF (10) |
AAC (100) |
974.3±62.3 |
991.7±2.5 |
114.3±11.7 |
186.7±14.3 |
68.0±0.0 |
|
S9-Mix |
With |
||||
Test Item (mg/plate) |
Base-pair substitution type |
Frameshift type |
|||
TA100 |
TA 102 |
TA 1535 |
TA98 |
TA1537 |
|
VC |
166.3±2.5 |
295.0±4.0 |
18.0±2.0 |
28.7±0.6 |
7.7±2.1 |
31.6 |
135.0±11.3 |
278.0±3.0 |
15.7±0.6 |
30.7±3.1 |
9.0±1.0 |
100 |
138.7±9.3 |
267.3±13.2 |
16.0±1.0 |
27.0±1.0 |
6.7±2.9 |
316 |
142.3±23.1 |
255.7±2.1 |
20.3±3.1 |
25.3±0.6 |
7.0±1.7 |
1000 |
126.3±14.5 |
270.0±26.9 |
15.7±0.6 |
25.7±6.0 |
7.3±2.5 |
3160 |
141.0±7.2 |
270.3±7.8 |
15.7±2.5 |
34.3±0.6 |
4.3±0.6 |
5000 |
32.3±3.5 |
93.3±2.1 |
7.3±1.2 |
8.3±0.6 |
1.7±0.6 |
PC (mg/plate) |
AAN (2) |
B[a]P (10) |
AAN (2) |
B[a]P (10) |
B[a]P (10) |
935.0±69.4 |
1026.0±72.5 |
133.0±24.3 |
185.0±6.0 |
64.3±0.6 |
VC = Vehicle control; PC = Positive control
NaN3= Sodium azide
2NF = 2-Nitrofluorene
MMC = Mitomycin C
AAC = 9-Aminoacridine
AAN = 2-Aminoanthracene
B[a]P = Benzo[a]pyrene
Results:
Concentration (µg/mL medium) |
Cloning Efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+06 Surviving Cells |
Mutation Factor |
1stExperiment – 3-hour exposure (-S9) |
||||
0 |
69.95 |
100 |
149.03 |
-- |
0.316 |
70.65 |
132 |
137.93 |
-11.10 |
1.0 |
79.28 |
71 |
126.89 |
-22.14 |
3.16 |
66.74 |
81 |
78.06 |
-70.97 |
10.0 |
30.43 |
12 |
161.68 |
12.65 |
MMS (0.013) |
39.53 |
40 |
542.88 |
393.85 |
1stExperiment – 3-hour exposure (+S9) |
||||
0 |
72.54 |
100 |
131.13 |
-- |
0.316 |
75.90 |
233 |
61.07 |
-70.06 |
1.0 |
73.75 |
216 |
58.98 |
-72.15 |
3.16 |
70.65 |
171 |
129.02 |
-2.11 |
10.0 |
45.98 |
17 |
178.01 |
46.88 |
3-MC (1.0) |
22.48 |
14 |
875.89 |
744.76 |
2ndExperiment – 24-hour exposure (-S9) |
||||
0 |
65.80 |
100 |
52.21 |
-- |
0.316 |
70.65 |
96 |
55.56 |
3.35 |
1.0 |
80.45 |
164 |
64.76 |
12.55 |
3.16 |
72.70 |
114 |
53.99 |
1.78 |
10.0 |
23.89 |
19 |
123.48 |
71.27 |
MMS (0.013) |
10.23 |
10 |
711.14 |
658.93 |
2ndExperiment – 3-hour exposure (+S9) |
||||
0 |
71.66 |
100 |
54.78 |
-- |
0.316 |
71.66 |
122 |
68.66 |
13.88 |
1.0 |
67.69 |
108 |
76.97 |
22.19 |
3.16 |
57.11 |
144 |
58.92 |
4.14 |
10.0 |
16.69 |
18 |
160.28 |
105.50 |
3-MC (1.0) |
42.03 |
28 |
824.53 |
769.75 |
0 = 0.05 M HCl
MMS = Methylmethanesulfonate
3-MC = 3-Methylcholanthrene
Results of Main Experiment
Test item |
Concentration |
CBPI |
Number of micronucleated cells/1000 binucleate cells |
µg/mL |
% |
||
Exposure period 4 h, fixation time 25 h, without S9 |
|||
0.05 M HCl |
-- |
1.45 |
7.5 |
Mitomycin C |
0.2 |
1.43 |
32.5 |
dizinc pyrophosphate |
6.25 |
1.51 |
9.5 |
12.5 |
1.60 |
8.0 |
|
25 |
1.22 |
5.5 |
|
50 |
1.08 |
cytotoxicity |
|
Exposure period 24 h, fixation time 45 h, without S9 |
|||
0.05 M HCl |
-- |
1.33 |
7.5 |
Colchicine |
0.01 |
1.31 |
34.0 |
dizinc pyrophosphate |
6.25 |
1.31 |
6.5a |
12.5 |
1.30 |
10.5a |
|
25 |
1.16 |
17.5a,b |
|
50 |
1.10 |
cytotoxicity |
|
CONFIRMATORY REPEAT Exposure period 24 h, fixation time 45 h, without S9 |
|||
0.05 M HCl |
-- |
1.41 |
5.0 |
Colchicine |
0.02 |
1.20 |
36.5 |
dizinc pyrophosphate |
10 |
1.38 |
5.5a |
15 |
1.35 |
13.0a,b |
|
25 |
1.19 |
21.5a,b |
|
35 |
1.09 |
cytotoxicity |
|
Exposure period 4 h, fixation time 25 h, with S9 |
|||
0.05 M HCl |
-- |
1.47 |
5.5 |
cyclophosphamide |
20 |
1.32 |
20.0 |
dizinc pyrophosphate |
6.25 |
1.42 |
8.5 |
12.5 |
1.34 |
6.5 |
|
25 |
1.23 |
8.5 |
|
50 |
1.10 |
13.0 (cytotoxicity) |
0.05 M HCl (vehicle/solvent control)
Mitomycin C, colchicine and cyclophosphamide (positive controls)
aDose-related increase in micronuclei up to 25µg/mL dizinc pyrophosphate/mL medium (Spearman’s correlation test: p-value of 0.0111)
bSignificantly greater than concurrent vehicle/solvent control mean.
In addition, several in vitro chromosome aberration assays are available for zinc sulfate and zinc chloride.
Litton, 1974: zinc sulfate: negative in human embryonic lung cells (WI-38)
Deknudt, 1978: zinc chloride: ambigous effects in human lymphocyte cultures
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
No in vivo data is available for dizinc pyrophosphate.
Based on the available data with different zinc compounds there is insufficient ground to classify zinc as genotoxic. There is no clear evidence from the available data that zinc is genotoxic in vivo.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- Summary of available data used for the endpoint assessment of the target substance
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks on result:
- other: Gocke, 1991, zinc sulfate
- Conclusions:
- Interpretation of results: inconclusive
- Executive summary:
As explained in the analogue justification, zinc is considered to be the toxic element. Therefore, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity potential.
Reference
In addition, several in vivo chromosome aberration and micronucleus assays are available for zinc sulfate and zinc chloride.
Gocke 1991: zinc sulfate: negative after oral treatment up to 86.3 mg/kg bw in mice
Litton, 1974: zinc sulfate: negative after oral treamtment up to 275 mg/kg bw in rats
Deknudt, 1979: zinc chloride: positive effects were observed with 0.5% zinc chloride orally daily through standard and low-calcium diet for one month
Gupta, 1991: zinc chloride: positive effects after intraperitoenal treatment up to 15 mg/kg bw in mice.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Gene mutations in bacteria
Mutagenic properties of dizinc pyrophosphate in bacteria were investigated according to OECD 471 with Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 (LPT, 2016). Dizinc pyrophosphate was examined in the Salmonella typhimurium strains in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was not soluble in any of the solvents recommended: water, dimethylsulfoxide (DMSO), ethanol or acetone. Dizinc pyrophosphate was completely soluble at 3.16 mg per mL 0.05 M HCl solution. Hence, dizinc pyrophosphate was suspended in 0.05 M HCl solution for concentrations of 1000, 3160 and 5000 μg per plate. For concentrations lower than 1000 μg/plate, the test substance was completely dissolved. The vehicle 0.05 M HCl solution served as the negative control. Six concentrations, 31.6, 100, 316, 1000, 3160, and 5000 μg dizinc pyrophosphate/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. Test substance precipitation was noted in both experiments, each carried out without and with metabolic activation, at concentrations of 3160 and 5000 μg/plate in all test strains. Cytotoxicity (reduction of the number of revertants by more than 50%) was noted at the top concentration of 5000 μg/plate in all test strains. No increase in revertant colony numbers as compared with control counts was observed for dizinc pyrophosphate, tested up to a cytotoxic concentration of 5000 μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively. Test substance precipitation was observed starting at 3160 μg/plate in all test strains with and without metabolic activation. The positive control substances showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. Under the conditions of the study, dizinc pyrophosphate was non-mutagenic in bacteria.
Cytogenicity
The clastogenic potential of dizinc pyrophosphate was assessed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals according to OECD 487 (LPT, 2016). The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied. The test item was not soluble in any of the solvents recommended: aqueous media, dimethylsulfoxide (DMSO), ethanol or acetone. However, dizinc pyrophosphate was completely soluble at 3.16 mg per mL 0.05 M HCl solution. For a concentration of 316 μg/mL medium, the test item was completely dissolved. This solution was then further diluted to the appropriate concentrations. The vehicle 0.05 M HCl solution served as the negative control. No correction factor was used. The concentrations employed were chosen based on the results of a cytotoxicity study and a preliminary experiment without and with metabolic activation. Hence, 50 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation. In addition, 35 μg/mL were employed as the top concentration for the repeat genotoxicity test without metabolic activation (24-hour exposure). In the main study cytotoxicity was noted in the experiments without and with metabolic activation at the top concentrations of 35 or 50 μg test substance/mL medium. Mitomycin C (at 0.2 μg/mL) and colchicine (at 0.01 or 0.02 μg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 μg/mL) in the presence of metabolic activation. In the test without metabolic activation (4-hour exposure) the micronucleus frequencies of cultures treated with the concentrations of 6.25, 12.5 or 25 μg dizinc pyrophosphate/mL medium in the absence of metabolic activation (4-hour exposure) ranged from 5.5 to 9.5 micronucleated cells per 1000 binucleated cells. At the top concentration of 50 μg/mL medium not enough binucleated cells could be evaluated due to cytotoxicity of the test substance. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Values of 8.0 and 9.5 micronucleated cells per 1000 binucleated cells were slightly above the upper limit of the 95% confidence interval of the historical background data at LPT. However, there was no dose-related increase in the number of micronuclei up to the concentration of 25 μg dizinc pyrophosphate/mL medium. Vehicle controls should give reproducibly low and consistent micronuclei frequencies. In this test a mean frequency of 7.5 micronucleated cells per 1000 binucleated cells was observed. The vehicle results were within the historical control ranges. However, the mean frequency of micronucleated cells in the experiment was slightly above the upper limit of the 95% confidence interval of the historical background data at LPT. However, the vehicle control in this study gave reproducibly low and consistent micronuclei frequencies without extreme outliers. Therefore, the slightly higher frequency in the vehicle group and treated groups was not judged to be biologically relevant. In the positive control cultures the mean micronucleus frequency was increased to 32.5 micronucleated cells per 1000 binucleate cells. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus. In the test without metabolic activation (24-hour exposure) the micronucleus frequencies of cultures treated with the concentrations of 6.25, 12.5 or 25 μg dizinc pyrophosphate/mL medium ranged from 6.5 to 17.5 micronucleated cells per 1000 binucleated cells. At the top concentration of 50 μg/mL medium not enough binucleated cells could be evaluated due to cytotoxicity of the test item. There was a dose-related increase in micronuclei up to the concentration of 25 μg dizinc pyrophosphate/mL medium (Spearman’s correlation test: p-value of 0.0111). The frequency of micronucleated cells was outside the distribution of the historical negative control data. The frequency of micronucleated cells exhibits a statistically significant increase compared with the concurrent negative control of 7.5 micronucleated cells per 1000 binucleated cells. This positive response of the test item was confirmed with a repeat experiment: The micronucleus frequencies of cultures treated with the concentrations of 10, 15 or 25 μg dizinc pyrophosphate/mL medium in the absence of metabolic activation (24-hour exposure) ranged from 5.5 to 21.5 micronucleated cells per 1000 binucleated cells. At the top concentration of 35 μg/mL medium not enough binucleated cells could be evaluated due to cytotoxicity of the test substance. There was a dose-related increase in micronuclei up to the concentration of 25 μg dizinc pyrophosphate/mL medium (Spearman’s correlation test: p-value of 0.0111). Vehicle controls should give reproducibly low and consistent micronuclei frequencies. A mean frequency of 7.5 micronucleated cells per 1000 binucleated cells was observed in the first test and a mean frequency of 5.0 micronucleuted cells per 1000 binucleated cells was observed in the repeat experiment. The vehicle results were within the historical control ranges. However, the mean frequency of micronucleated cells in the first test was slightly above the upper limit of the 95% confidence interval of the historical background data at LPT. However, the vehicle control in this study gave reproducibly low and consistent micronuclei frequencies without extreme outliers. Therefore, the slightly higher frequency in the vehicle group was not judged to be biologically relevant. In the positive control cultures the mean micronucleus frequency was increased to 34.0 or 36.5 micronucleated cells per 1000 binucleate cells. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus. In the test with metabolic activation (4-hour exposure) the micronucleus frequencies of cultures treated with the concentrations of 6.25, 12.5 or 25 μg dizinc pyrophosphate/mL medium (4-h exposure) in the presence of metabolic activation ranged from 6.5 to 8.5 micronucleated cells per 1000 binucleated cells. The marginally high micronuclei frequency of 13.0 micronucleated cells per 1000 binucleated cells was calculated by 3 or 4 micronucleated cells per 240 or 305 binucleated cells, respectively, at the pronounced cytotoxic concentration of 50 μg/mL medium. Although the value of 8.5 micronucleated cells per 1000 binucleated cells was marginally above the upper limit of the 95% confidence interval of the historical background data at LPT, there was no dose-related increase in micronuclei up to the top concentration of 25 μg dizinc pyrophosphate/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronuclei frequencies. In this test a mean frequency of 5.5 micronucleated cells per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges. In the positive control culture the micronucleus frequency was increased to 20.0 micronucleated cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.
Under the experimental conditions of this study, the test substance induced chromosome breaks and/or gain or loss in human lymphocytes. For this study possible false positive results can be excluded as no relevant changes in pH or osmolality were noted for any of the tested concentrations of dizinc pyrophosphate. A statistically significant concentration-related increase in the frequency of micronucleated cells was observed in the non-cytotoxic concentrations and in concentrations that produced at a maximum of 55 ± 5% cytotoxicity only.
Gene mutations in mammalian cells
Mutagenic properties on mammalian cells of dizinc pyrophosphate were determined in in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats according to OECD 490 (LPT, 2016). The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays. The test item was not soluble in any of the solvents recommended. Dizinc pyrophosphate was completely soluble at 2 mg per mL 0.05 M HCl solution. Hence, 40, 20 or 6.32 mg dizinc pyrophosphate were suspended in 1 mL 0.05 M HCl solution. These suspensions were diluted 1:20 with culture medium to obtain a final concentration. For a concentration of 100 μg/mL medium, the test substance was completely dissolved. This solution was then further diluted to the appropriate concentrations. The vehicle 0.05 M HCl solution served as the negative control. Based on the results of the preliminary study five concentrations of 0.316, 1.0, 3.16, 10.0 and 31.6 μg dizinc pyrophosphate per mL medium for the experiments without and with metabolic activation with a 3-hour exposure and 0.1, 0.316, 1.0, 3.16 and 10.0 μg for the experiment without metabolic activation with a 24-hour exposure were employed in the mutagenicity tests. Methylmethanesulfonate (MMS) was used as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (3-MC) in the presence of exogenous metabolic activation. In the main study, pronounced cytotoxicity (decreased survival) was noted at concentrations of 10.0 and 31.6 μg dizinc pyrophosphate/mL in the absence and presence of metabolic activation (3-hour exposure) and at 3.16 and 10.0 μg/mL in the absence of metabolic activation (24-hour exposure). The top concentrations of 31.6 μg dizinc pyrophosphate/mL medium or 10.0 μg/mL, respectively, were excluded from evaluation of mutation frequencies because of the pronounced cytotoxicity with a relative total growth (RTG) of <10. The values of mutation frequencies of the negative controls ranged from 49.01 to 149.03 per 106cloneable cells in the experiments without metabolic activation and from 52.82 to 143.31 per 106cloneable cells in the experiments with metabolic activation and, hence, were all well within the historical data-range with the exception of the value (24-hour exposure without S9) which was marginally below the historical data-range. The mutation frequencies of the cultures treated with dizinc pyrophosphate ranged from 78.06 to 161.68 per 106cloneable cells (3 hours exposure) and from 53.99 to 123.48 per 106cloneable cells (24 hours exposure) in the experiments without metabolic activation and from 58.98 to 178.01 per 106cloneable cells (3 hours exposure, first assay) and from 58.92 to 160.28 per 106cloneable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation. In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.12 to 1.25 for dizinc pyrophosphate-treated cells and from 0.17 to 0.80 for the negative controls. The positive controls MMS and 3-MC caused pronounced increases in the mutation frequency. In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.68 to 2.25 in the case of MMS. Under the experimental conditions of this study, dizinc pyrophosphate was negative with respect to the mutant frequency in the L5178Y TK + /- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, dizinc pyrophosphate also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that dizinc pyrophosphate, tested up to the cytotoxic concentrations, did neither induce mutations nor have any chromosomal aberration potential.
Conclusion: Based on the three test results on in vitro genotoxicity dizinc pyrophosphate showed positive outcome in the cytogenicity test. However, it has to be pointed out, that the published genotoxic test results for zinc salts are non-conclusive and variable. Various zinc salts showed negative as well as positive results when tested in several in vitro genotoxic test systems. There is also no clear experimental or epidemiological evidence for a direct carcinogenic action of zinc, albeit that zinc deficiency or supplementation may influence carcinogenesis given that promoting and inhibiting actions have been reported (SIDS). Since dizinc pyrophosphate showed positive results in the cytogenetic assay the available data on the clastogenic potential of zinc salts was further evaluated. The zinc ion is considered to be the toxicologically relevant element and thus a read across to zinc sulfate and zinc chloride is reliable. A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).
In vitro:
A chromosome aberration test was performed in human dental pulp cells (D824) with zinc chloride with and without metabolic activation (Someya, 2008). D824 cells have the capability of accumulating calcium in vitro and generating dentin-like tissue when transplanted into immunocompromised mice. In addition, odontoblast-like cells which are differentiated from D824 cells are observed in the connective tissue compartment near the dentin-like tissue. D824 cells are comparable or more sensitive to induction of chromosome aberrations when compared with human lymphocytes as well as mouse and hamster cells. The cells were treated with zinc chloride for 3 h. After treatment, cells were washed and subsequently incubated for a further 27 h. Three hours before the end of incubation, colcemid was administered and metaphase chromosomes were prepared. The sampling time for preparing metaphase chromosomes was approximately 1.4 times the normal cellcycle length for the beginning of treatment with test agents. In the second experiment, cells were treated with zinc chloride for 30 h. Three hours before the end of treatment, colcemid was administered and metaphase chromosomes were prepared. As the test agents showed negative responses in the above experiments, additional experiments were performed under the exogenous metabolic activation system. Cells were treated with test agents for 2 h in the presence of 5% postmitochondrial supernatant (PMS). After treatment, cells were washed then incubated for 28 h. Three hours before the end of incubation, colcemid was added. The aberrations scored were chromatid gaps, chromosome gaps, chromatid breaks, chromosome breaks, chromatid exchanges, dicentric chromosomes, ring chromosomes, and fragmentations. Metaphases were also scored for the induction of polyploidy and endoreduplication, but these numerical aberrations were not taken into account as chromosome aberrations in the present study. Under the conditions of this test zinc chloride did not show any chromosome aberrations with and without metabolic activation whereas the positive control showed a positive response in this assay. No statistically significant increase in the percentage of cells with polyploidy or endoreduplication was observed in cells treated with zinc chloride.
A further chromosome aberration test with zinc chloride is available (Deknudt, 1978). This test was conducted on human lymphocyte cultures to determine the mutagenic potential of zinc chloride. The concentration of zinc chloride inhibiting mitotic activity was found to be 0.003 M. Three subtoxic doses i.e., 0.0015, 0.0003 and 0.00003 M were taken for the study. Human lymphocytes were obtained from a healthy donor and cultured for 48 or 72 h in Ham's F 10 medium. The test substance was added to 48 and 72 h cultures at 0 and 24 h after initiation. Chromosome preparations were prepared and 100 well-spread metaphases from each culture were evaluated for the presence of numerical and structural aberrations. Chromosome aberrations (dicentric chromosomes) were observed at the lowest concentration of 0.00003 M of the test substance. However, the results were found to be insignificant when compared to only controls in chi-square analysis and thus the results are considered ambiguous.
A further chromosome aberration test with zinc sulfate is available (Litton, 1974). This test was conducted on human embryonic lung cultures (Wi-38) to determine the mutagenic potential of zinc sulfate. The concentration of zinc sulfate inhibiting mitotic activity was found to be 25µg/mL. Three subtoxic doses i.e., 0.1, 1.0 and 10 µg/mL were taken for the study. Human embryonic lung cultures were treated with 0.1, 1.0, and 10.0 µg/mL zinc sulfate for 24 to 48 hours until mitosis took place. Chromosome preparations were prepared and the anaphase of 100 cells from each culture were evaluated for the presence of chromosome aberrations. The negative control group showed no aberrations as did the 0.1 µg/mL test group. Both 1.0 and 10 µg/mL exhibited 1% of the cells with bridges. The positive control group contained 15% of the cells with aberrations showing the validity of the test. Thus, zinc sulfate did not produce a detectable significant aberration in the anaphase chromosomes of human tissue culture cells in this study.
In vivo:
A micronucleus test on mouse bone marrow was conducted to evaluate the mutagenic potential of the zinc sulfate (Gocke, 1981). Male and female NMRI mice were used for the study. Feed and water were provided ad libitum. 4 animals (2 male, 2 female) were used in each treatment and control groups. 86.3, 57.5 and 28.8 mg/kg doses were administered i.p. at 0 and 24 h. Bone marrow smears were prepared at 30 h. 1000 polychromatic erythrocytes were scored per mouse. Significance was calculated according to the Kastenbaum-Bowman tables. Under the test conditions, test material was found to be non-mutagenic.
A study was conducted to determine the chromosomal aberration inducing capacity of zinc chlorid using mice bone marrow cells (Deknudt, 1978). No guideline or GLP compliance was documented in the study report. C57BI male mice were exposed to 0.5% zinc chlorid orally daily through standard and low-calcium diet for one month. The metaphase bone marrow cells were obtained, exposed to hypotonic solution and fixed. The cells were then spread on slides and stained using lacto-orcein. 50 well-spread metaphase cells from each animal (a total of 500 from each group) were analysed for chromosomal aberrations and the results were evaluated using chi-square method. Body weights of mice were significantly reduced on treatment with the test material in standard diet as well as low-calcium diet alone. However, the effect was more pronounced with a combined treatment of zinc chlorid and low calcium diet. Serum calcium was reduced in animals on a low calcium diet and this effect was accentuated to 9.76 ± 0.29, by intoxication with the test substance 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 significantly increased in mice kept on a low calcium diet plus test material. Thus, the zinc chloride caused severe chromosomal anomalies, particularly in animals kept on a low calcium diet under the conditions of the test.
Another study was conducted to determine the clastogenicity of zinc chloride using mammalian bone marrow chromosomal aberration test (Gupta, 1991). Swiss albino mice were exposed to the test substance intraperitoneally at a concentration of 7.5, 10 and 15 mg/kg bw once (acute treatment) and 2 and 3 mg/kg bw every alternate day for 8, 16 and 24 d (chronic treatment). Animals were sacrificed by cervical dislocation and bone marrow cells preparations were made and analysed. Significant (p<0.05) increase in the frequencies of chromosomal aberrations were observed at all concentrations used following acute and chronic treatment. But the control group used is not entirely appropriate for chronic evaluation as the animals were treated with isotonic saline and sacrificed after 24 hours. Thus, zinc chloride is a potent clastogen after acute exposure.
A study was conducted to determine the chromosomal aberration inducing capacity of zinc sulfate using rat bone marrow cells (Litton, 1974). No guideline or GLP compliance was documented in the study report. Male Sprague Dawley rats were exposed to the test substance by gavage at a concentration of 2.75, 27.5 and 275 mg/kg bw once (acute treatment) and for 5 consecutive days (subactue treatment). In the acute study bone marrow was prepared after 6, 24 and 48 hours respectively. In the subacute study bone marrow was prepared 6 hours after last treatment. Animals were sacrificed by CO2 and bone marrow cells preparations were made and analysed. Zinc sulfate produced no detectable significant aberrations of the bone marrow metaphase chromosomes in any dose or treatment duration. The acute treatment with triethylenemelamine as positive control showed significant aberrations of the bone marrow metaphase chromosmes thus showing the validity of the test. Thus, zinc sulfate showed no clastogen potential in this study.
Final conclusion:
Genotoxicity data on dizinc pyrophosphate are available for gene mutation in bacterial and mammalian cells and a cytogenicity in mammalian cells. Dizinc pyrophosphate did not show any potential for gene mutation in bacterial and mammalian cells respectively. Dizinc pyrophosphate induced micronuclei in human lymphocytes. Data on other zinc compounds have been used based on the assumption that after intake the biological activities of the zinc compounds are determined by the zinc cation. The available data indicate that the genotoxicity results vary widely. Conflicting results have been found, even in the same test systems. Overall, the results of the in vitro tests indicate that zinc has genotoxic potential in vitro based on positive results in mammalian test systems for chromosomal aberrations. In vivo, increases in chromosomal aberrations were found in calcium-deficient mice exposed via the diet as well as in mice with normal calcium status when dosed intraperitoneally. In mice also negative results were obtained and even at higher dose levels. Rats tested negative for chromosomal aberrations after oral dosing. Conflicting results are also observed in vivo, however mainly negative in vivo results were observed. Based on the available data there is insufficient ground to classify zinc as genotoxic. There is no clear evidence from the available data that zinc is genotoxic in vivo. This conclusion is in line with those achieved by other regulatory reviews of the genotoxicity of zinc compounds (WHO, 2001; EU RAR, 2004, MAK, 2009). Hence, no classification and labelling for mutagenicity are required for dizinc pyrophosphate.
References:
EU RAR: European Union Risk assessment report: zinc oxide. 2004 (https://echa.europa.eu/documents/10162/cc20582a-d359-4722-8cb6-42f1736dc820)
MAK- und BAT-Werte-Liste 2009: Maximale Arbeitsplatzkonzentrationen und Biologische Arbeitsstofftoleranzwerte. Hrsg.: Senatskommsion zur Prüfung gesundheitsgefährdender Arbeitsstoffe der Deutschen Forschungsgemeinschaft. Weinheim: Wiley-VCH 2009
WHO 2001: Environmental Health Criteria 221: Zinc (http://www.who.int/ipcs/publications/ehc/ehc_221/en/)
Justification for classification or non-classification
Based on the available data with the test substance itself and read across on genetic toxicity indicate that dizinc pyrophosphate does not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC.
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