Registration Dossier
Registration Dossier
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EC number: 238-735-6 | CAS number: 14691-80-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
A weight of evidence approach is used on the test substance and analagous substances to fulfil the Annex VII, Section 8.4.1 endpoint: in vitro gene mutation study in bacteria.
A key study performed on an analogous substance is available for the Annex VIII Section 8.4.2 in vitro cytogenicity in mammalian cells and Section 8.4.3 in vitro gene mutation study in mammalian cells endpoint.
A range of supporting studies are available for all endpoints, including the Annex VIII Section 8.4.2 in vitro cytogenicity study in mammalian cells or in vitro micronucleus study.
None of the studies suggest the substance is mutagenic.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- no data
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- significant methodological deficiencies
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- AMES study conducted using 3 strains of S. typhimurium only, positive control substances differ from those recommended in the guideline, only one dose level investigated
- GLP compliance:
- no
- Remarks:
- Study predates GLP
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- not applicable
- Species / strain / cell type:
- S. typhimurium, other: TA 1535, TA 1537, TA 1538
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- yeast, other: Saccharomyces cerevisiae D4
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Metabolic activation preparations derived from the tissues of ICR random bred male mice, Sprague-Dawley adult male rats and Macaca mulatta adult male primates
- Test concentrations with justification for top dose:
- Plate test (S. typhimurium): 0.1%
Suspension tests (S. typhimurium): 1.13, 2,25%
Suspension tests (S. cerevisiae): 0.05, 0.1% - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: no data
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Used in non-activation assays with tester strains TA 1535 and S. cerevisiae D4
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: quinacrine mustard
- Remarks:
- Used in a non-activation assay with tester strain TA 1537
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- Used in a non-activation assay with tester strain TA 1538
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: dimethylnitrosamine
- Remarks:
- Used in an activation assay with tester strain TA 1535 and S. cerevisiae D4
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- Used in an activation assay with tester strains TA 1537 and TA 1538
- Details on test system and experimental conditions:
- Two types of assay were conducted; plate tests and suspension tests.
1. PLATE TESTS (bacteria only):
METHOD OF APPLICATION:
Nonactivation studies: plate incorporation
Activation studies: in suspension
DURATION
- Exposure duration: 4 days at 37°C
2. SUSPENSION TESTS (bacteria and yeast):
METHOD OF APPLICATION: in suspension
Nonactivation tests:
DURATION
- Exposure duration: 4 hours at 30°C (yeast assays), 1 hour at 37°C (bacterial assay)
- Selection time (if incubation with a selection agent): 48 hours at 37°C (bacterial assay), 3-5 days at 30°C
Activation tests:
DURATION
- Exposure duration: 4 hours at 30°C (yeast assays), 1 hour at 37°C (bacterial assay)
DETERMINATION OF CYTOTOXICITY
- Method: Each chemical was tested for survival against the specific indicator strains over a range of doses to determine 50% survival dose. - Evaluation criteria:
- no data
- Statistics:
- No data
- Species / strain:
- S. typhimurium, other: TA 1353, TA 1357, TA 1358
- 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:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- yeast, other: Saccharomyces cerevisiae D4
- 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:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test substance was soluble at the treatment concentration employed in this study. All tests were conducted in an aqueous environment.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test date for toxicity determinations: September 15, 1974
The 50% survival level was determined for bacterial and yeast indicator organisms by conducting survival curves with the test compound at the following concentrations (w/w or v/v): For D4: 1, 2, 3, 4, 5. For S. typhimurium: 0.001, 0.005, 0.01, 0.05, 0.1%
-Concentrations of the test compound used in mutagenicity tests:
BACTERIA:
- Plate tests: 0.1%
-1/4 50% survival: 0.05%
-1/2 50% survival: 0.1%
YEAST:
- Plate tests: --
-1/4 50% survival: 1.13%
-1/2 50% survival: 2.25% - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Tetrasodium pyrophosphate did not exhibit genetic activity in any of the assays employed and is considered to be non-mutagenic under the conditions of this study.
This study is submitted to provide a weight of evidence to support the conclusion that sodium and potassium pyrophosphates are unlikely to produce a positive genotoxic response. - 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:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium, other: TA 1353, TA 1357, TA 1358
- 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:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- yeast, other: Saccharomyces cerevisiae D4
- 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:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test substance was soluble at the treatment concentration employed in this study. All tests were conducted in an aqueous environment.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test date for toxicity determinations: September 15, 1974
The 50% survival level was determined for bacterial and yeast indicator organisms by conducting survival curves with the test compound at the following concentrations (w/w or v/v): For D4: 1, 2, 3, 4, 5. For S. typhimurium: 0.001, 0.005, 0.01, 0.05, 0.1%
-Concentrations of the test compound used in mutagenicity tests:
BACTERIA:
- Plate tests: 0.1%
-1/4 50% survival: 0.05%
-1/2 50% survival: 0.1%
YEAST:
- Plate tests: --
-1/4 50% survival: 1.13%
-1/2 50% survival: 2.25% - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with tetrasodium pyrophosphate.
- Executive summary:
Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with tetrasodium pyrophosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium hydrogen diphosphate and tetra sodium pyrophosphate are unlikely to lead to differences in the genotoxicity
that are higher than the typical experimental error of the test method.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The mutation test was carried out in accordance with the preincubation procedure described by Ames et al.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Not applicable
- Species / strain / cell type:
- S. typhimurium, other: TA 97 and TA 102
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 0.1, 0.5, 1, 5, 10 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Used with tester strain TA 97 without S9 mix Migrated to IUCLID6: (50 µg)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 mins
- Exposure duration: no data
DETERMINATION OF CYTOTOXICITY
No data - Evaluation criteria:
- No data
- Statistics:
- Kruskal-Wallis test and regression analysis
- Species / strain:
- S. typhimurium, other: TA 97 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- No data
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The authors conclude that sodium pyrophosphate is non-mutagenic under the conditions of this study.
This study is submitted to provide a weight of evidence to support the conclusion that sodium and potassium pyrophosphates are unlikely to produce a positive genotoxic response. - 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:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium, other: TA 97 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- No data
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with tetrasodium pyrophosphate.
- Executive summary:
Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with tetrasodium pyrophosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium hydrogen diphosphate and tetra sodium pyrophosphate are unlikely to lead to differences in the genotoxicity
that are higher than the typical experimental error of the test method.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- significant methodological deficiencies
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- No postive control data
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- not specified
- Remarks:
- Study pre-dates GLP
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Not applicable
- Species / strain / cell type:
- S. typhimurium, other: TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with
- Metabolic activation system:
- Rat liver S-9
- Test concentrations with justification for top dose:
- Maximum dose: 10.0 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Phosphate buffer
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- exposed to appropriate solvent or untreated
- True negative controls:
- not specified
- Positive controls:
- no
- Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 2 days at 37 °C- Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). A negative result indicates that no significant increases in the numbers of revertant colonies were detected in any S. typhimurium strains at the maximum dose.- Statistics:
- No data
- Species / strain:
- S. typhimurium, other: TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Additional information on results:
- No data
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Disodium dihydrogen pyrophosphate is considered to be non-mutagenic in strains of S.typhimurium, under the conditions of this assay.
This study is submitted to provide a weight of evidence to support the conclusion that sodium and potassium pyrophosphates are unlikely to produce a positive genotoxic response. - 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:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium, other: TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Additional information on results:
- No data
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with sodium acid pyrophosphate.
- Executive summary:
Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with sodium acid pyrophosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium hydrogen diphosphate and sodium acid pyrophosphate are unlikely to lead to differences in the genotoxicity
that are higher than the typical experimental error of the test method.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- significant methodological deficiencies
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Study does not cover all tester strains recommended in the guideline. Positive control substances differ from those recommended in the guideline
- Principles of method if other than guideline:
- In addition to the bacterial studies, the same assay was also performed on Saccharomyces cerevisiae, strain: D4.
- GLP compliance:
- not specified
- Remarks:
- Study predates GLP
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Not applicable
- Species / strain / cell type:
- S. typhimurium, other: TA 1535, TA 1537, TA 1538 TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- yeast, other: Saccharomyces cerevisiae, D4
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 homogenate prepared from Sprague-Dawley adult male rat liver induced by Aroclor 1254 5-days prior to kill.
- Test concentrations with justification for top dose:
- 0.001, 0.01, 0.1, 1.0 and 5 μL per plate
- Vehicle / solvent:
- - Vehicle/solvent used: distilled water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: methylnitrosoguanidine (10 µg/plate)
- Remarks:
- Used with tester strains TA 1535, TA 100 and S. cerevisiae; D4, in assays without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: quinacrine mustard (10 µg/plate)
- Remarks:
- Used with tester strains TA 1537, in assays without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- Used with tester strains TA 1538 and TA 98, in assays without metabolic activation Migrated to IUCLID6: 100 µg/plate
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-anthramine (100 µg/plate)
- Remarks:
- Used with tester strains TA 1535 and TA 100, in assays with metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 8-aminoquinoline (100 µg/plate)
- Remarks:
- Used with tester strains TA 1537, in assays with metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- Remarks:
- Used with tester strains TA 1538 and TA 98, in assays with metabolic activation Migrated to IUCLID6: 100 µg/plate
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: DMNA (100 µMol/plate)
- Remarks:
- Used with tester strains S. cerevisiae; D4, in assays with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: plate incorporation
DURATION
- Exposure duration: 48 hr at 37°C
- Expression time (cells in growth medium): overnight - Evaluation criteria:
- The criteria used to determine positive effects are inherently subjective and are based on historical data. Most data sets were evaluated using the following criteria:
- STRAINS TA 1353, TA-1357 and TA 1358
If the solvent control value is within the normal range, a chemical that produces a positive dose response over 3 concentrations with the lowest increase equal to 2x the solvent control value is considered to be mutagenic (positive result).
- STRAINS TA 98, TA 100 AND D4:
If the solvent control value is within the normal range, a chemical that produces a positive response of 3 concentrations with the highest increase equal to 2x the solvent value (TA 100) and 2-3x the solvent control value (TA 98 and D4) is considered to be mutagenic (positive result). The dose-response increase should start at approximately the solvent control value. - Statistics:
- no data
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level.
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- yeast, other: Saccharomyces cerevisiae, D4
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level.
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY: The substance was tested over a series of concentrations. The dose range employed in the study was below a concentration that demonstrated any toxic effect.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The test compound MCTR-257 did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered not-mutagenic under these test conditions.
This study is submitted to provide a weight of evidence to support the conclusion that sodium and potassium pyrophosphates are unlikely to produce a positive genotoxic response. - 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:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level.
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- yeast, other: Saccharomyces cerevisiae, D4
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level.
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY: The substance was tested over a series of concentrations. The dose range employed in the study was below a concentration that demonstrated any toxic effect.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with tetrapotassium pyrophosphate.
- Executive summary:
Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with tetrapotassium pyrophosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium hydrogen diphosphate and tetrapotassium pyrophosphate are unlikely to lead to differences in the genotoxicity
that are higher than the typical experimental error of the test method.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- not specified
- Remarks:
- Study published in the literature. No information on GLP is given.
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9: induced with Aroclor 1254 or a combination of phenobarbitone and β-naphthoflavone.
- Test concentrations with justification for top dose:
- 0, 301.25, 602.5, 1,205, 2,410, 4,820 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: No data - Untreated negative controls:
- yes
- Remarks:
- 0 µg/plate
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Untreated negative controls:
- yes
- Remarks:
- 0 µg/plate
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Untreated negative controls:
- yes
- Remarks:
- 0 µg/plate
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (2-AF)
- Untreated negative controls:
- yes
- Remarks:
- 0 µg/plate
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- Remarks:
- Migrated to IUCLID6: (2-AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); without metabolic activation
preincubation; with metabolic activation
DURATION
- Preincubation period: 20 minutes or more
- Exposure duration: 48-72 hrs at 37°C
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity detected by a reduction in the number of reverting colonies, a clearing or diminution of the background lawn, or the degree of survival of treated cultures. - Evaluation criteria:
- Number of revertant colonies per plate were counted. The mutant frequency was expressed as the quotient of the number of revertant colonies over the number of colonies in the negative control. A mutagenic potential of a test item was assumed if the mutant frequency is 2.0 or higher.
- Statistics:
- No data
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- 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:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Tetrasodium pyrophosphate was found to be non-genotoxic in an AMES test both with and without metabolic activation.
This study is submitted to provide a weight of evidence to support the conclusion that sodium and potassium pyrophosphates are unlikely to produce a positive genotoxic response. - 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:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- 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:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with tetrasodium pyrophosphate.
- Executive summary:
Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with tetrasodium pyrophosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium hydrogen diphosphate and tetrasodium pyrophosphate are unlikely to lead to differences in the genotoxicity
that are higher than the typical experimental error of the test method.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- no data
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- significant methodological deficiencies
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- In vitro mutagenicity in bacteria: Method as described by Ames et al, 1973, Prot. Nat. Acad. Sci. U.S.A. 70, 2281-2285
In vitro miotic recombination assay in yeast: The yeast wasexposed to the appropriate concentration of test compound for 4 hours and samples were plated. The mitotic recombination frequency is expressed as sectors per 105 survivors. This was compared with a negative control. - GLP compliance:
- no
- Remarks:
- Study predates GLP
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- no data
- Species / strain / cell type:
- S. typhimurium, other: TA 1535, TA 1536, TA 1537 and TA 1538
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- Saccharomyces cerevisiae
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- no data
- Test concentrations with justification for top dose:
- Bacterial assay: no data
Yeast assay: 5.0% w/v - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: no data
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- EMS used in yeast assay
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 1-fluorenamine
- Remarks:
- 1-fluorenamine was used for metabolic activation in the bacterial assay
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: no data
DURATION
- Preincubation period: no data
- Exposure duration: 4 hours
- Expression time (cells in growth medium): no data
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): no data - Evaluation criteria:
- no data
- Statistics:
- no data
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1536, TA 1537 and TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- Saccharomyces cerevisiae
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- a concentration of 5.0% w/v was used
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- no data
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In the in vitro assays, sodium acid pyrophosphate was not mutagenic to S. typhimurium either in the presence or absence of the metabolic activation system. At a concentration of 5% sodium acid pyrophosphate was not toxic to nor did it increase the mitotic recombination frequency of S. cerevisiae D3.
This study is submitted to provide a weight of evidence to support the conclusion that sodium and potassium pyrophosphates are unlikely to produce a positive genotoxic response. - 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:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1536, TA 1537 and TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- Saccharomyces cerevisiae
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- a concentration of 5.0% w/v was used
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- no data
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with sodium acid pyrophosphate.
- Executive summary:
Trisodium hydrogen diphosphate is estimated to be non-genotoxic as found in the source study performed with sodium acid pyrophosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium hydrogen diphosphate and sodium acid pyrophosphate are unlikely to lead to differences in the genotoxicity
that are higher than the typical experimental error of the test method.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 September 2016 - 07 September 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- adopted September 26, 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Date of Inspection: 16-19 April 2013 Date on Certificate: 14 May 2014
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1531481
- Expiration date of the lot/batch: 19/07/2016
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in a tightly closed container and stored in a cool, dry and well-ventilated place.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test item was completely dissolved in highly purified water
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Fresh preparations of the test item were prepared on the day of the experiment and used for the treatment in all experimental parts. - Species / strain / cell type:
- lymphocytes: human peripheral lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Human peripheral blood
- Suitability of cells: cells identified in OECD guideline.
- Sex, age and number of blood donors if applicable: Healthy non-smoking male of female individuals (18-35 years). No known recent exposures to genotoxic chemicals or radiation.
- Whether whole blood or separated lymphocytes were used if applicable:
Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Cytochalasin B
The concentration used for this assay was 5 μg/mL. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- In the preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Sodium dihydrogen orthophosphate/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium.
Hence, 2000 µg/mL medium were employed as top concentration in the main study for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Highly purified water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- 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
- Preincubation period: 48 hours
- Exposure duration: 4hours and 24 hours at +37°C
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours
STAIN (for cytogenetic assays): 10% Giemsa
NUMBER OF REPLICATIONS: 2 (main study), 1 (preliminary test)
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide and left to air-dry.
NUMBER OF CELLS EVALUATED: The micronucleus frequencies were analysed in at least 2000 binucleate cells per concentration
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: relative total growth (relative increase in cell counts, RI); cytotoxicity = 100% - RI [%] - Evaluation criteria:
- If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei, it is classified as a positive result.
Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response. - Key result
- Species / strain:
- lymphocytes: human peripheral lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolarity : No relevant changes in pH or osmolality of the test item formulations at concentrations of 3.16 to 2000 µg/mL medium were noted
- Water solubility: Soluble in water
RANGE-FINDING/SCREENING STUDIES: . In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Sodium dihydrogen orthophosphate/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium. Hence, 2000 µg/mL were employed as the top concentration for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure).
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Cytokinesis block proliferation index ranged between 1.26-1.48 for the test substance
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see tables below
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (micronucleus frequencies per 1000 cells):
Mitomycin C:
Mean: 46.9
SD: 35
range 17-137
Colchicine:
Mean: 25.9
SD: 9.5
range 15-63
cyclophosphamide:
Mean: 44.0
SD: 37.7
range 14-158
- Negative (solvent/vehicle) historical control data:
Without metabolic activation
Untreated control
mean 6.6
SD 2.9
range 2.0 - 17
95% Confidence interval 5.9 - 7.3
Vehicle control
mean 6.3
SD 3.1
range 2.0 - 18
95% Confidence interval 5.7 - 6.8
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI or RI in the case of the cytokinesis-block method - Conclusions:
- Under the present test conditions, sodium dihydrogenorthophosphate tested up to a concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
- Executive summary:
Test samples of Sodium dihydrogen orthophosphate were assayed 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.
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 completely dissolved in highly purified water. The vehicle highly purified water served as the negative control.
The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 μg Sodium dihydrogen orthophosphate/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 μg/mL medium.
Hence, 2000 μg/mL were employed as the top concentration for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure).
No signs of cytotoxicity were noted in the main study up to the top concentration of 2000 μg Sodium dihydrogen orthophosphate/mL medium in the experiments without and with metabolic activation.
Mitomycin C (at 0.2 μg/mL) and colchicine (at 0.02 μg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 μg/mL) in the presence of metabolic activation.
Tests without metabolic activation (4- and 24-hour exposure)
The micronucleus frequencies of cultures treated with the concentrations of 125, 250, 500, 1000 and 2000 or 250, 500, 1000 and 2000 μg Sodium dihydrogen orthophosphate/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 3.0 to 4.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 μg/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 micronucleus frequencies. In this test a frequency of 3.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure was observed. The vehicle result was within the historical control ranges.
In the positive control cultures the micronucleus frequencies were increased to 37.5 or 53.5 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.
Test with metabolic activation (4-hour exposure)
The micronucleus frequencies of cultures treated with the concentrations of 125, 250, 500, 1000 and 2000 μg Sodium dihydrogen orthophosphate/ml medium (4-h exposure) in the presence of metabolic activation ranged from 2.5 to 4.5 micronucleated cells per 1000 bi nucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 μg/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 micronucleus frequencies. In this test a mean frequency of 3.5 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 27 .0 micronucleated cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.
Conclusion
Under the present test conditions, Sodium dihydrogen orthophosphate tested up to a concentration of 2000 μg/ml medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
The results for the vehicle controls were within historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- lymphocytes: human peripheral lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolarity : No relevant changes in pH or osmolality of the test item formulations at concentrations of 3.16 to 2000 µg/mL medium were noted
- Water solubility: Soluble in water
RANGE-FINDING/SCREENING STUDIES: . In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Sodium dihydrogen orthophosphate/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium. Hence, 2000 µg/mL were employed as the top concentration for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure).
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Cytokinesis block proliferation index ranged between 1.26-1.48 for the test substance
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see tables below
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (micronucleus frequencies per 1000 cells):
Mitomycin C:
Mean: 46.9
SD: 35
range 17-137
Colchicine:
Mean: 25.9
SD: 9.5
range 15-63
cyclophosphamide:
Mean: 44.0
SD: 37.7
range 14-158
- Negative (solvent/vehicle) historical control data:
Without metabolic activation
Untreated control
mean 6.6
SD 2.9
range 2.0 - 17
95% Confidence interval 5.9 - 7.3
Vehicle control
mean 6.3
SD 3.1
range 2.0 - 18
95% Confidence interval 5.7 - 6.8
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI or RI in the case of the cytokinesis-block method - Conclusions:
- Under the present test conditions, sodium dihydrogenorthophosphate tested up to a concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
- Executive summary:
Test samples of Sodium dihydrogen orthophosphate were assayed 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.
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 completely dissolved in highly purified water. The vehicle highly purified water served as the negative control.
The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 μg Sodium dihydrogen orthophosphate/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 μg/mL medium.
Hence, 2000 μg/mL were employed as the top concentration for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure).
No signs of cytotoxicity were noted in the main study up to the top concentration of 2000 μg Sodium dihydrogen orthophosphate/mL medium in the experiments without and with metabolic activation.
Mitomycin C (at 0.2 μg/mL) and colchicine (at 0.02 μg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 μg/mL) in the presence of metabolic activation.
Tests without metabolic activation (4- and 24-hour exposure)
The micronucleus frequencies of cultures treated with the concentrations of 125, 250, 500, 1000 and 2000 or 250, 500, 1000 and 2000 μg Sodium dihydrogen orthophosphate/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 3.0 to 4.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 μg/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 micronucleus frequencies. In this test a frequency of 3.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure was observed. The vehicle result was within the historical control ranges.
In the positive control cultures the micronucleus frequencies were increased to 37.5 or 53.5 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.
Test with metabolic activation (4-hour exposure)
The micronucleus frequencies of cultures treated with the concentrations of 125, 250, 500, 1000 and 2000 μg Sodium dihydrogen orthophosphate/ml medium (4-h exposure) in the presence of metabolic activation ranged from 2.5 to 4.5 micronucleated cells per 1000 bi nucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 μg/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 micronucleus frequencies. In this test a mean frequency of 3.5 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 27 .0 micronucleated cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.
Conclusion
Under the present test conditions, Sodium dihydrogen orthophosphate tested up to a concentration of 2000 μg/ml medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
The results for the vehicle controls were within historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 07 September 2016 to 21 November 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Date of Inspection: 16-19 April 2013 Date of Signature on Certificate: 14 May 2014
- Type of assay:
- other: gene mutation in mammalian cells
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No: 1541481
- Expiration date of the lot/batch: November 2018
- Purity test date: 99.8 %
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in a tightly closed container and stored at a dry, cool and well-ventilated place
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Completely dissolved in highly purified water
FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution
OTHER SPECIFICS: - Target gene:
- Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: The indicator cell used for this study was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-). The particular clone (3.7.2C) used in this assay is isolated by ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA.
- Methods for maintenance in cell culture if applicable: Master stock cultures were maintained in liquid nitrogen.Laboratory cultures were periodically checked for karyotype stability and the absence of mycoplasma contamination by culturing methods. To reduce the background mutant frequency (spontaneous frequency) of TK / mutants to a level as low as possible, cell cultures were exposed to conditions that select against the TK / phenotype (exposure to aminopterin or methotrexate).
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cells used during the experimental studies were maintained in growth medium RPMI 1640 with glutamaxTM medium supplemented with Pluronic® F68 , gentamycin , amphotericin B1 and horse serum1 (10% by volume).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver enzymes (S9 fraction) and an energy producing system comprising nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and glucose-6-phosphate.
- Test concentrations with justification for top dose:
- Based on the results of the preliminary study five concentrations of 125, 250, 500, 1000 and 2000 µg for the experiments without and with metabolic activation were employed in the mutagenicity tests.
A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation. A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000, and 2000 µg PRAYPHOS™ MSP FG/FG GR/mL medium were tested for cytotoxicity. Cytotoxicity (decreased survival) was noted at a concentration of 1000 or 2000 µg/mL in the presence of metabolic activation (3-hour exposure). No changes in pH or osmolality were noted in the test item formulations compared to the control.
Hence, in the main study the highest concentration employed was 2000 µg PRAYPHOS™ MSP FG/FG GR/mL medium in the experiments without and with metabolic activation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Vehicle (purified water) treatment groups were used as the negative control
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Vehicle treatment groups (purified water) were used as the negative control
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (490) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.
One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at five dose levels, in duplicate, together with vehicle (water) and positive controls. The exposure groups used were as follows: 3 hour exposures both with and without metabolic activation. A repeat experiment was used with 3 hour exposure for the metabolic activation groups and 24 hours for the without metabolic activation groups.
The dose range of test material was selected following the results of a preliminary toxicity test and was 125 to 2000 µg/ml for all exposure groups.
In the preliminary study, cytotoxicity (decreased survival) was noted at a concentration of 1000 or 2000 µg/mL in the presence of metabolic activation (3-hour exposure). Hence, in the main study the highest concentration employed was 2000 µg PRAYPHOS™ MSP FG/FG GR/mL medium in the experiments without and with metabolic activation. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the exposure groups. - Evaluation criteria:
- Please see Interpretation of Results in "Any other information and methods incl. tables" section. As this section will not accommodate the required information.
- Statistics:
- Please see Interpretation of Results in "Any other information and methods incl. tables" section. As this section will not accommodate the required information.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- non-mutagenic
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In preliminary experiment, cytotoxicity was noted at a concentration of 1000 or 2000 ug/mL in the presence of metabolic activation (3-hour exposure)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Results
A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation.A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000, and 2000 µg sodium dihydrogenorthophosphate/mL medium were tested for cytotoxicity. Cytotoxicity (decreased survival) was noted at a concentration of 1000 or 2000 µg/mL in the presence of metabolic activation (3-hour exposure). For details, seeTable 1. No changes in pH or osmolality were noted in the test item formulations compared to the control (seeText table 7-1 'pH values and osmolality').
Hence, in the main study the highest concentration employed was 2000 µg sodium dihydrogenorthophosphate/mL medium in the experiments without and with metabolic activation.
Methylmethanesulfonate (0.013 or 0.012 µg/mL for a 3- and 24-hour exposure, respectively) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (1.0 µg/mL) in the presence of exogenous metabolic activation.
In the main study, cytotoxicity (decreased relative total growth) was noted in the first experiment with metabolic activation at concentrations of 1000 and 2000 µg sodium dihydrogenorthophosphate/mL medium and at 2000 µg/mL medium in the second experiment with S9. No signs of cytotoxicity were noted in the experiments without metabolic activation.
The negative controls had mutation frequencies of 63.89 or 137.93 per 106 clonable cells in the experiments without metabolic activation (3- or 24-hour exposure, respectively, for details seeTable 3a) and 55.47 or 120.71 per 106 clonable cells in the experiments with metabolic activation (for details see Table 3b) and, hence, were all well within the historical data-range.
The mutation frequencies of the cultures treated with sodium dihydrogenorthophosphate ranged from 57.17 to 74.73 per 106 clonable cells (3-hour exposure) and from 84.33 to 164.59 per 106clonable cells (24-hour exposure) in the experiments without metabolic activation (for details seeTable 3a). In the experiments with metabolic activation, mutation frequencies ranged from 63.82 to 86.21 per 106 clonable cells (3-hour exposure, first assay) and from 50.23 to 155.35 per 106 clonable cells (3-hour exposure, second assay) (for details, seeTable 3b). These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable 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 0.40to 1.31 for sodium dihydrogenorthophosphate-treated cells and ratios of 0.42 to 0.94for the negative controls (see Table 4a and Table 4b).
The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3‑MC) caused pronounced increases in the mutation frequency of 474.25 and 652.55 per 106clonable cells in the case of MMS (for details seeTable 3a) and of 387.42 and 489.98 per 106clonable cells in the case of 3-MC (for details seeTable 3b). All positive controls showed an increase in the small colony MF of at least 150 x 10-6above that seen in the concurrent solvent control and an absolute increase in total mutation frequency of at least 300 x 10-6. Furthermore, the mean relative total growth (RTG) for the positive controls was greater than or equal to 10%. Hence, the acceptance criteria were met.
The calculations of suspension growth of the negative controls were in the acceptance criteria range between 8 and 32 following 3-hour treatments or between 32 and 180 following 24-hour treatments (for details see Table 2a and Table 2b). The mean cloning efficiencies (CE = PEx 100) of the negative controls from the Mutation Experiments were between the range 65% to 120% (see Table 3a and Table 3b). Hence, the acceptance criteria described in were met.
The mutation frequency and the colony size ratio of the positive controls and negative controls without and with metabolic activation for the last 11 experiments (background data, not audited by the QAU-department) are attached.
The pH and osmolality data of the negative control and of all test item formulations in the medium were determined in the first preliminary test - see Text table 7-1 below in 'Any other information on results incl. tables.'
No relevant changes in pH or osmolality of the test item formulations at concentrations of 10.0 to 2000 µg/mL medium compared to the negative control were noted. - Conclusions:
- Under the present test conditions, sodium dihydrogenorthophosphate, tested up to a concentration of 2000 µg/mL medium, the recommended maximum concentration by the guideline, in two independent experiments 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, sodium dihydrogenorthophosphate also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that sodium dihydrogenorthophosphate, tested up to a concentration of 2000 µg/mL medium, neither induced mutations nor had any chromosomal aberration potential. - Executive summary:
In order to investigate the mutagenic potential on mammalian cells, the sodium dihydrogenorthophosphatewas assayed in a gene mutation assay 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. 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.
Sodium dihydrogenorthophosphate was completely dissolved in highly purified water. The vehicle highly purified water served as the negative control.
A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation. A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000, and 2000 µg sodium dihydrogenorthophosphate/mL medium were tested for cytotoxicity. Cytotoxicity (decreased survival) was noted at a concentration of 1000 or 2000 µg/mL in the presence of metabolic activation (3-hour exposure). No changes in pH or osmolality were noted in the test item formulations compared to the control.
Hence, in the main study the highest concentration employed was 2000 µg sodium dihydrogenorthophosphate/mL medium in the experiments without and with metabolic activation.
Methylmethanesulfonate (0.013 or 0.012 µg/mL for a 3- and 24-hour exposure, respectively) was employed as a positive control in the absence of exogenous metabolic activation and 3‑Methylcholanthrene (1.0 µg/mL) in the presence of exogenous metabolic activation.
In the main study, cytotoxicity (decreased relative total growth) was noted in the first experiment with metabolic activation at concentrations of 1000 and 2000 µg sodium dihydrogenorthophosphate/mL medium and at 2000 µg/mL medium in the second experiment with S9. No signs of cytotoxicity were noted in the experiments without metabolic activation.
The negative controls had mutation frequencies of 63.89 or 137.93 per 106clonable cells in the experiments without metabolic activation(3- or 24-hour exposure, respectively,and 55.47or 120.71 per 106clonable cells in the experiments with metabolic activationand, hence, were all well within the historical data-range.
The mutation frequencies of the cultures treated with sodium dihydrogenorthophosphate ranged from 57.17 to 74.73 per 106clonable cells (3-hour exposure) and from 84.33 to 164.59 per 106clonable cells (24-hour exposure) in the experiments without metabolic activation. In the experiments with metabolic activation, mutation frequencies ranged from 63.82 to 86.21 per 106clonable cells (3-hour exposure, first assay) and from 50.23 to 155.35 per 106clonable cells (3-hour exposure, second assay). 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 0.40 to 1.31 for sodium dihydrogenorthophosphate -treated cells and ratios of 0.42 to 0.94 for the negative controls.
The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3‑MC) caused pronounced increases in the mutation frequency of 474.25 and 652.55 per 106clonable cells in the case of MMS and of 387.42 and 489.98 per 106clonable cells in the case of 3-MC. As the increase in the small colony mutation frequency was at least 150 x 10-6above the concurrent negative control, anabsolute increase in totalmutation frequency was at least 300 x 10-6 for the positive controls and the mean relative total growth (RTG) greater than or equal to 10%, the acceptance criteria for the positive controls were met.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category
(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.
It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.
3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.
4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- non-mutagenic
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In preliminary experiment, cytotoxicity was noted at a concentration of 1000 or 2000 ug/mL in the presence of metabolic activation (3-hour exposure)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Results
A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation.A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000, and 2000 µg sodium dihydrogenorthophosphate/mL medium were tested for cytotoxicity. Cytotoxicity (decreased survival) was noted at a concentration of 1000 or 2000 µg/mL in the presence of metabolic activation (3-hour exposure). For details, seeTable 1. No changes in pH or osmolality were noted in the test item formulations compared to the control (seeText table 7-1 'pH values and osmolality').
Hence, in the main study the highest concentration employed was 2000 µg sodium dihydrogenorthophosphate/mL medium in the experiments without and with metabolic activation.
Methylmethanesulfonate (0.013 or 0.012 µg/mL for a 3- and 24-hour exposure, respectively) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (1.0 µg/mL) in the presence of exogenous metabolic activation.
In the main study, cytotoxicity (decreased relative total growth) was noted in the first experiment with metabolic activation at concentrations of 1000 and 2000 µg sodium dihydrogenorthophosphate/mL medium and at 2000 µg/mL medium in the second experiment with S9. No signs of cytotoxicity were noted in the experiments without metabolic activation.
The negative controls had mutation frequencies of 63.89 or 137.93 per 106 clonable cells in the experiments without metabolic activation (3- or 24-hour exposure, respectively, for details seeTable 3a) and 55.47 or 120.71 per 106 clonable cells in the experiments with metabolic activation (for details see Table 3b) and, hence, were all well within the historical data-range.
The mutation frequencies of the cultures treated with sodium dihydrogenorthophosphate ranged from 57.17 to 74.73 per 106 clonable cells (3-hour exposure) and from 84.33 to 164.59 per 106clonable cells (24-hour exposure) in the experiments without metabolic activation (for details seeTable 3a). In the experiments with metabolic activation, mutation frequencies ranged from 63.82 to 86.21 per 106 clonable cells (3-hour exposure, first assay) and from 50.23 to 155.35 per 106 clonable cells (3-hour exposure, second assay) (for details, seeTable 3b). These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable 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 0.40to 1.31 for sodium dihydrogenorthophosphate-treated cells and ratios of 0.42 to 0.94for the negative controls (see Table 4a and Table 4b).
The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3‑MC) caused pronounced increases in the mutation frequency of 474.25 and 652.55 per 106clonable cells in the case of MMS (for details seeTable 3a) and of 387.42 and 489.98 per 106clonable cells in the case of 3-MC (for details seeTable 3b). All positive controls showed an increase in the small colony MF of at least 150 x 10-6above that seen in the concurrent solvent control and an absolute increase in total mutation frequency of at least 300 x 10-6. Furthermore, the mean relative total growth (RTG) for the positive controls was greater than or equal to 10%. Hence, the acceptance criteria were met.
The calculations of suspension growth of the negative controls were in the acceptance criteria range between 8 and 32 following 3-hour treatments or between 32 and 180 following 24-hour treatments (for details see Table 2a and Table 2b). The mean cloning efficiencies (CE = PEx 100) of the negative controls from the Mutation Experiments were between the range 65% to 120% (see Table 3a and Table 3b). Hence, the acceptance criteria described in were met.
The mutation frequency and the colony size ratio of the positive controls and negative controls without and with metabolic activation for the last 11 experiments (background data, not audited by the QAU-department) are attached.
The pH and osmolality data of the negative control and of all test item formulations in the medium were determined in the first preliminary test - see Text table 7-1 below in 'Any other information on results incl. tables.'
No relevant changes in pH or osmolality of the test item formulations at concentrations of 10.0 to 2000 µg/mL medium compared to the negative control were noted. - Conclusions:
- Trisodium hydrogen diphosphate is estimated to be non-genotoxic in a L5178Y TK +/- mammalian cell mutagenicity test as found in the source study performed with sodium dihydrogenorthophosphate.
- Executive summary:
Trisodium hydrogen diphosphate is estimated to be non-genotoxic in a L5178Y TK +/- mammalian cell mutagenicity test as found in the source study performed with sodium dihydrogenorthophosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium hydrogen diphosphate and sodium dihydrogenorthophosphate
are unlikely to lead to differences in the genotoxicity that are higher than the typical experimental error of the test method.
Referenceopen allclose all
PLATE TESTS:
Table 2: Results of plate tests
TEST |
SPECIES |
TISSUE |
REVERTANTS/PLATES |
|||||
TA 1535 |
TA 1537 |
TA 1538 |
||||||
1 |
2 |
1 |
2 |
1 |
2 |
|||
1. Nonactivation |
||||||||
Positive control –(0.5 mL) |
- |
- |
>103 |
>103 |
|
|
|
|
Positive control – QM (0.25 mg) |
- |
- |
|
|
>102 |
>102 |
|
|
Positive control – NF (0.25 mg) |
- |
- |
|
|
|
|
>102 |
>102 |
Solvent control - saline |
|
|
2 |
1 |
2 |
4 |
|
|
Solvent control – DMSO (<10%) |
|
|
|
|
|
|
5 |
1 |
Test compound – 1 (0.1%) |
- |
- |
2 |
2 |
6 |
8 |
1 |
3 |
2. Activation |
||||||||
Positive control - DMNA |
Mouse |
Liver |
>103 |
>103 |
|
|
|
|
Positive control – AAF |
Liver |
|
|
44 |
43 |
>102 |
>102 |
|
Positive control – DMNA |
Lung |
2 |
4 |
|
|
|
|
|
Positive control – AAF |
Lung |
|
|
9 |
3 |
13 |
8 |
|
Positive control – DMNA |
Testes |
1 |
5 |
|
|
|
|
|
Positive control – AAF |
Testes |
|
|
6 |
10 |
3 |
3 |
|
Solvent control –DMNA |
- |
3 |
0 |
|
|
|
|
|
Solvent control – AAF |
- |
|
|
10 |
5 |
1 |
0 |
|
Solvent control – saline |
- |
1 |
1 |
|
|
|
|
|
Solvent control –DMSO (<10%) |
- |
|
|
12 |
10 |
6 |
7 |
|
Test compound (0.1%) |
Liver |
2 |
0 |
7 |
9 |
17 |
12 |
|
Test compound (0.1%) |
Lung |
1 |
2 |
8 |
5 |
8 |
7 |
|
Test compound (0.1%) |
Testes |
0 |
0 |
9 |
4 |
12 |
15 |
|
Positive control – DMNA (25 µmoles) |
Rat |
Liver |
>102 |
>102 |
|
|
|
|
Positive control – AAF (1.25 mg) |
Liver |
|
|
41 |
30 |
>102 |
>102 |
|
Positive control – DMNA (25 µmoles) |
Lung |
1 |
0 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
Lung |
|
|
7 |
10 |
5 |
0 |
|
Positive control – DMNA (25 µmoles) |
Testes |
3 |
0 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
Testes |
|
|
14 |
17 |
10 |
3 |
|
Positive control – DMNA (25 µmoles) |
- |
3 |
0 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
- |
|
|
10 |
5 |
1 |
0 |
|
Solvent control – saline |
- |
1 |
1 |
|
|
|
|
|
Solvent control –DMSO (<10%) |
- |
|
|
12 |
10 |
6 |
7 |
|
Test compound (0.1%) |
Liver |
1 |
0 |
9 |
4 |
18 |
5 |
|
Test compound (0.1%) |
Lung |
1 |
0 |
7 |
9 |
3 |
2 |
|
Test compound (0.1%) |
Testes |
0 |
2 |
4 |
11 |
5 |
6 |
|
Positive control – DMNA (25 µmoles) |
Monkey |
Liver |
>102 |
>102 |
|
|
|
|
Positive control – AAF (1.25 mg) |
Liver |
|
|
32 |
46 |
>102 |
>102 |
|
Positive control – DMNA (25 µmoles) |
Lung |
0 |
4 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
Lung |
|
|
20 |
13 |
2 |
4 |
|
Positive control – DMNA (25 µmoles) |
Testes |
1 |
1 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
Testes |
|
|
10 |
11 |
3 |
7 |
|
Positive control – DMNA (25 µmoles) |
- |
3 |
0 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
- |
|
|
10 |
5 |
1 |
0 |
|
Solvent control – saline |
- |
1 |
1 |
|
|
|
|
|
Solvent control –DMSO (<10%) |
- |
|
|
12 |
10 |
6 |
7 |
|
Test compound |
Liver |
2 |
1 |
12 |
8 |
5 |
8 |
|
Test compound |
Lung |
1 |
0 |
7 |
7 |
8 |
5 |
|
Test compound |
Testes |
0 |
0 |
5 |
5 |
7 |
8 |
|
SUSPENSION TESTS:
Table 3. Results of suspension tests with and without activation
Test |
Species |
Organ |
Salmonella reversion frequencies |
D4 conversion frequencies (x 10-5) |
|||
TA 1535 |
TA 1537 |
TA 1538 |
Ade+ |
Trp+ |
|||
Nonactivation |
|||||||
Positive control |
- |
- |
1104.27 |
115.80 |
48.98 |
89.60 |
112.73 |
Negative control |
- |
- |
1.46 |
11.81 |
10.61 |
6.55 |
4.37 |
High dose |
- |
- |
2.12 |
10.29 |
7.39 |
6.44 |
4.93 |
Low dose |
- |
- |
1.26 |
9.11 |
10.11 |
5.86 |
3.86 |
Activation |
|||||||
Solvent control |
- |
- |
2.42 |
6.62 |
5.82 |
5.54 |
4.62 |
Chemical control |
- |
- |
2.22 |
8.49 |
7.07 |
6.16 |
3.54 |
Positive control |
Mouse |
Liver |
731.67 |
15.10 |
29.84 |
8.20 |
8.83 |
Positive control |
Mouse |
Lung |
6.59 |
4.95 |
7.11 |
6.20 |
4.57 |
Positive control |
Mouse |
Testes |
5.62 |
2.17 |
8.59 |
6.09 |
5.41 |
High dose |
Mouse |
Liver |
0.97 |
11.07 |
7.29 |
7.17 |
6.05 |
Low dose |
Liver |
2.94 |
6.79 |
8.74 |
6.24 |
6.86 |
|
High dose |
Lung |
1.70 |
6.12 |
4.72 |
6.11 |
5.65 |
|
Low dose |
Lung |
1.00 |
4.82 |
8.00 |
7.71 |
7.13 |
|
High dose |
Testes |
0.69 |
3.07 |
5.78 |
7.19 |
4.04 |
|
Low dose |
Testes |
0.49 |
3.02 |
2.54 |
6.70 |
3.87 |
|
Solvent control |
- |
- |
2.67 |
7.52 |
9.55 |
3.40 |
2.92 |
Chemical control |
- |
- |
3.33 |
17.09 |
3.88 |
2.71 |
2.50 |
Positive control |
Rat |
Liver |
245.70 |
29.39 |
42.80 |
9.42 |
10.27 |
Positive control |
Rat |
Lung |
0.97 |
10.17 |
5.90 |
3.91 |
3.55 |
Positive control |
Rat |
Testes |
1.68 |
8.83 |
6.08 |
0.23 |
2.02 |
High dose |
Rat |
Liver |
1.33 |
9.83 |
6.91 |
-- |
3.01 |
Low dose |
Liver |
2.05 |
8.14 |
5.73 |
3.78 |
2.81 |
|
High dose |
Lung |
1.23 |
6.89 |
5.69 |
6.52 |
3.42 |
|
Low dose |
Lung |
0.71 |
6.94 |
7.69 |
5.72 |
2.63 |
|
High dose |
Testes |
1.49 |
6.94 |
1.80 |
6.68 |
4.20 |
|
Low dose |
Testes |
0.55 |
2.32* |
3.47 |
4.40 |
2.45 |
|
Solvent control |
- |
- |
2.49 |
16.19 |
13.85 |
2.86 |
1.33 |
Chemical control |
- |
- |
4.27 |
18.27 |
9.18 |
4.06 |
1.43 |
Positive control |
Monkey |
Liver |
206.49 |
26.72 |
30.32 |
9.81 |
2.82 |
Positive control |
Monkey |
Lung |
2.49 |
8.64 |
13.79 |
3.94 |
3.62 |
Positive control |
Monkey |
Testes |
2.79 |
6.91 |
8.71 |
7.10 |
3.84 |
High dose |
Monkey |
Liver |
1.60 |
15.98 |
9.51 |
5.51 |
3.06 |
Low dose |
Liver |
1.97 |
11.72 |
9.09 |
5.41 |
5.84 |
|
High dose |
Lung |
1.68 |
14.89 |
11.45 |
2.56 |
3.84 |
|
Low dose |
Lung |
2.00 |
12.41 |
8.68 |
5.75 |
2.03 |
|
High dose |
Testes |
1.22 |
8.22 |
3.26 |
2.72 |
1.09 |
|
Low dose |
Testes |
3.81 |
5.79 |
3.56 |
5.00 |
2.50 |
|
* Data from repeat tests
PLATE TESTS:
Table 2: Results of plate tests
TEST |
SPECIES |
TISSUE |
REVERTANTS/PLATES |
|||||
TA 1535 |
TA 1537 |
TA 1538 |
||||||
1 |
2 |
1 |
2 |
1 |
2 |
|||
1. Nonactivation |
||||||||
Positive control –(0.5 mL) |
- |
- |
>103 |
>103 |
|
|
|
|
Positive control – QM (0.25 mg) |
- |
- |
|
|
>102 |
>102 |
|
|
Positive control – NF (0.25 mg) |
- |
- |
|
|
|
|
>102 |
>102 |
Solvent control - saline |
|
|
2 |
1 |
2 |
4 |
|
|
Solvent control – DMSO (<10%) |
|
|
|
|
|
|
5 |
1 |
Test compound – 1 (0.1%) |
- |
- |
2 |
2 |
6 |
8 |
1 |
3 |
2. Activation |
||||||||
Positive control - DMNA |
Mouse |
Liver |
>103 |
>103 |
|
|
|
|
Positive control – AAF |
Liver |
|
|
44 |
43 |
>102 |
>102 |
|
Positive control – DMNA |
Lung |
2 |
4 |
|
|
|
|
|
Positive control – AAF |
Lung |
|
|
9 |
3 |
13 |
8 |
|
Positive control – DMNA |
Testes |
1 |
5 |
|
|
|
|
|
Positive control – AAF |
Testes |
|
|
6 |
10 |
3 |
3 |
|
Solvent control –DMNA |
- |
3 |
0 |
|
|
|
|
|
Solvent control – AAF |
- |
|
|
10 |
5 |
1 |
0 |
|
Solvent control – saline |
- |
1 |
1 |
|
|
|
|
|
Solvent control –DMSO (<10%) |
- |
|
|
12 |
10 |
6 |
7 |
|
Test compound (0.1%) |
Liver |
2 |
0 |
7 |
9 |
17 |
12 |
|
Test compound (0.1%) |
Lung |
1 |
2 |
8 |
5 |
8 |
7 |
|
Test compound (0.1%) |
Testes |
0 |
0 |
9 |
4 |
12 |
15 |
|
Positive control – DMNA (25 µmoles) |
Rat |
Liver |
>102 |
>102 |
|
|
|
|
Positive control – AAF (1.25 mg) |
Liver |
|
|
41 |
30 |
>102 |
>102 |
|
Positive control – DMNA (25 µmoles) |
Lung |
1 |
0 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
Lung |
|
|
7 |
10 |
5 |
0 |
|
Positive control – DMNA (25 µmoles) |
Testes |
3 |
0 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
Testes |
|
|
14 |
17 |
10 |
3 |
|
Positive control – DMNA (25 µmoles) |
- |
3 |
0 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
- |
|
|
10 |
5 |
1 |
0 |
|
Solvent control – saline |
- |
1 |
1 |
|
|
|
|
|
Solvent control –DMSO (<10%) |
- |
|
|
12 |
10 |
6 |
7 |
|
Test compound (0.1%) |
Liver |
1 |
0 |
9 |
4 |
18 |
5 |
|
Test compound (0.1%) |
Lung |
1 |
0 |
7 |
9 |
3 |
2 |
|
Test compound (0.1%) |
Testes |
0 |
2 |
4 |
11 |
5 |
6 |
|
Positive control – DMNA (25 µmoles) |
Monkey |
Liver |
>102 |
>102 |
|
|
|
|
Positive control – AAF (1.25 mg) |
Liver |
|
|
32 |
46 |
>102 |
>102 |
|
Positive control – DMNA (25 µmoles) |
Lung |
0 |
4 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
Lung |
|
|
20 |
13 |
2 |
4 |
|
Positive control – DMNA (25 µmoles) |
Testes |
1 |
1 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
Testes |
|
|
10 |
11 |
3 |
7 |
|
Positive control – DMNA (25 µmoles) |
- |
3 |
0 |
|
|
|
|
|
Positive control – AAF (1.25 mg) |
- |
|
|
10 |
5 |
1 |
0 |
|
Solvent control – saline |
- |
1 |
1 |
|
|
|
|
|
Solvent control –DMSO (<10%) |
- |
|
|
12 |
10 |
6 |
7 |
|
Test compound |
Liver |
2 |
1 |
12 |
8 |
5 |
8 |
|
Test compound |
Lung |
1 |
0 |
7 |
7 |
8 |
5 |
|
Test compound |
Testes |
0 |
0 |
5 |
5 |
7 |
8 |
|
SUSPENSION TESTS:
Table 3. Results of suspension tests with and without activation
Test |
Species |
Organ |
Salmonella reversion frequencies |
D4 conversion frequencies (x 10-5) |
|||
TA 1535 |
TA 1537 |
TA 1538 |
Ade+ |
Trp+ |
|||
Nonactivation |
|||||||
Positive control |
- |
- |
1104.27 |
115.80 |
48.98 |
89.60 |
112.73 |
Negative control |
- |
- |
1.46 |
11.81 |
10.61 |
6.55 |
4.37 |
High dose |
- |
- |
2.12 |
10.29 |
7.39 |
6.44 |
4.93 |
Low dose |
- |
- |
1.26 |
9.11 |
10.11 |
5.86 |
3.86 |
Activation |
|||||||
Solvent control |
- |
- |
2.42 |
6.62 |
5.82 |
5.54 |
4.62 |
Chemical control |
- |
- |
2.22 |
8.49 |
7.07 |
6.16 |
3.54 |
Positive control |
Mouse |
Liver |
731.67 |
15.10 |
29.84 |
8.20 |
8.83 |
Positive control |
Mouse |
Lung |
6.59 |
4.95 |
7.11 |
6.20 |
4.57 |
Positive control |
Mouse |
Testes |
5.62 |
2.17 |
8.59 |
6.09 |
5.41 |
High dose |
Mouse |
Liver |
0.97 |
11.07 |
7.29 |
7.17 |
6.05 |
Low dose |
Liver |
2.94 |
6.79 |
8.74 |
6.24 |
6.86 |
|
High dose |
Lung |
1.70 |
6.12 |
4.72 |
6.11 |
5.65 |
|
Low dose |
Lung |
1.00 |
4.82 |
8.00 |
7.71 |
7.13 |
|
High dose |
Testes |
0.69 |
3.07 |
5.78 |
7.19 |
4.04 |
|
Low dose |
Testes |
0.49 |
3.02 |
2.54 |
6.70 |
3.87 |
|
Solvent control |
- |
- |
2.67 |
7.52 |
9.55 |
3.40 |
2.92 |
Chemical control |
- |
- |
3.33 |
17.09 |
3.88 |
2.71 |
2.50 |
Positive control |
Rat |
Liver |
245.70 |
29.39 |
42.80 |
9.42 |
10.27 |
Positive control |
Rat |
Lung |
0.97 |
10.17 |
5.90 |
3.91 |
3.55 |
Positive control |
Rat |
Testes |
1.68 |
8.83 |
6.08 |
0.23 |
2.02 |
High dose |
Rat |
Liver |
1.33 |
9.83 |
6.91 |
-- |
3.01 |
Low dose |
Liver |
2.05 |
8.14 |
5.73 |
3.78 |
2.81 |
|
High dose |
Lung |
1.23 |
6.89 |
5.69 |
6.52 |
3.42 |
|
Low dose |
Lung |
0.71 |
6.94 |
7.69 |
5.72 |
2.63 |
|
High dose |
Testes |
1.49 |
6.94 |
1.80 |
6.68 |
4.20 |
|
Low dose |
Testes |
0.55 |
2.32* |
3.47 |
4.40 |
2.45 |
|
Solvent control |
- |
- |
2.49 |
16.19 |
13.85 |
2.86 |
1.33 |
Chemical control |
- |
- |
4.27 |
18.27 |
9.18 |
4.06 |
1.43 |
Positive control |
Monkey |
Liver |
206.49 |
26.72 |
30.32 |
9.81 |
2.82 |
Positive control |
Monkey |
Lung |
2.49 |
8.64 |
13.79 |
3.94 |
3.62 |
Positive control |
Monkey |
Testes |
2.79 |
6.91 |
8.71 |
7.10 |
3.84 |
High dose |
Monkey |
Liver |
1.60 |
15.98 |
9.51 |
5.51 |
3.06 |
Low dose |
Liver |
1.97 |
11.72 |
9.09 |
5.41 |
5.84 |
|
High dose |
Lung |
1.68 |
14.89 |
11.45 |
2.56 |
3.84 |
|
Low dose |
Lung |
2.00 |
12.41 |
8.68 |
5.75 |
2.03 |
|
High dose |
Testes |
1.22 |
8.22 |
3.26 |
2.72 |
1.09 |
|
Low dose |
Testes |
3.81 |
5.79 |
3.56 |
5.00 |
2.50 |
|
* Data from repeat tests
Table 1: Positive control data
Substance |
Solvent |
Dose (µg/plate) |
No. of revertants / plate (mean of 3 plates) |
|||
TA 97 |
TA 102 |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
|||
9-Aminoacridine |
DMSO |
50 µg |
627 ± 2131 |
|
|
|
Mitomycin C |
DMSO |
0.5 µg |
|
|
2438 ± 236 |
|
2-Aminoanthracene |
DMSO |
5 µg |
|
2104 ± 656 |
|
1144 ± 226 |
1. mean and standard deviation (n=19)
Table 2: Results of an AMES study conducted on sodium pyrophosphate:
Substance |
Solvent |
Dose (mg/plate) |
No. of revertant / plate (mean of 3 plates) |
|||
TA 97 |
TA 102 |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
|||
Sodium pyrophosphate |
DW |
10 |
122 |
187 |
334 |
328 |
5 |
125 |
195 |
348 |
333 |
||
1 |
114 |
205 |
334 |
489 |
||
0.5 |
130 |
207 |
342 |
476 |
||
0.1 |
150 |
202 |
341 |
500 |
||
0 |
151 |
184 |
340 |
496 |
||
Table 1: Positive control data
Substance |
Solvent |
Dose (µg/plate) |
No. of revertants / plate (mean of 3 plates) |
|||
TA 97 |
TA 102 |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
|||
9-Aminoacridine |
DMSO |
50 µg |
627 ± 2131 |
|
|
|
Mitomycin C |
DMSO |
0.5 µg |
|
|
2438 ± 236 |
|
2-Aminoanthracene |
DMSO |
5 µg |
|
2104 ± 656 |
|
1144 ± 226 |
1. mean and standard deviation (n=19)
Table 2: Results of an AMES study conducted on sodium pyrophosphate:
Substance |
Solvent |
Dose (mg/plate) |
No. of revertant / plate (mean of 3 plates) |
|||
TA 97 |
TA 102 |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
|||
Sodium pyrophosphate |
DW |
10 |
122 |
187 |
334 |
328 |
5 |
125 |
195 |
348 |
333 |
||
1 |
114 |
205 |
334 |
489 |
||
0.5 |
130 |
207 |
342 |
476 |
||
0.1 |
150 |
202 |
341 |
500 |
||
0 |
151 |
184 |
340 |
496 |
||
Table 1: Results as presented in study:
Additive |
Max dose (mg/plate) |
Result |
Disodium dihydrogen pyrophosphate |
10.0 |
Negative |
Table 1: Results as presented in study:
Additive |
Max dose (mg/plate) |
Result |
Disodium dihydrogen pyrophosphate |
10.0 |
Negative |
Table 2. Results of anstudy on MCTR-257
Test |
Dosage (μL) |
Revertants per plate |
|||||||||||
TA-1353 |
TA-1357 |
TA-1358 |
TA-98 |
TA-100 |
D4* |
||||||||
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
||
Non-activation |
|||||||||||||
Solvent control |
- |
11 |
|
10 |
|
11 |
|
30 |
|
154 |
|
68 |
|
Positive control |
** |
236 |
|
901 |
|
408 |
|
721 |
|
530 |
|
>1000 |
|
Test compound |
0.001 |
15 |
|
5 |
|
11 |
|
20 |
|
191 |
|
48 |
|
0.01 |
8 |
|
13 |
|
8 |
|
24 |
|
203 |
|
55 |
|
|
0.1 |
25 |
|
6 |
|
5 |
|
19 |
|
212 |
|
54 |
|
|
1.0 |
19 |
|
9 |
|
5 |
|
21 |
|
198 |
|
44 |
|
|
5.0 |
12 |
|
10 |
|
5 |
|
20 |
|
210 |
|
39 |
|
|
Activation |
|||||||||||||
Solvent control |
- |
13 |
|
16 |
|
16 |
|
43 |
|
124 |
|
35 |
|
Positive control |
*** |
242 |
|
208 |
|
428 |
|
650 |
|
>1000 |
|
82 |
|
Test compound |
0.001 |
17 |
|
23 |
|
20 |
|
45 |
|
162 |
|
50 |
|
0.01 |
19 |
|
21 |
|
22 |
|
41 |
|
170 |
|
61 |
|
|
0.1 |
16 |
|
15 |
|
23 |
|
48 |
|
170 |
|
58 |
|
|
1.0 |
14 |
|
23 |
|
18 |
|
40 |
|
154 |
|
52 |
|
|
5.0 |
16 |
|
6 |
|
21 |
|
39 |
|
179 |
|
52 |
|
|
* TRY+ convertants per plate.
**
TA-1535 |
MNNG |
10 μg/plate |
TA-1537 |
QM |
10 μg/plate |
TA-1538 |
NF |
100 μg/plate |
TA-98 |
NF |
100 μg/plate |
TA-100 |
MNNG |
10 μg/plate |
D4 |
MNNG |
10 μg/plate |
***
TA-1535 |
ANTH |
100 μg/plate |
TA-1537 |
AMQ |
100 μg/plate |
TA-1538 |
AAF |
100 μg/plate |
TA-98 |
AAF |
100 μg/plate |
TA-100 |
ANTH |
100 μg/plate |
D4 |
DMNA |
100 micromoles/plate |
Table 2. Results of anstudy on MCTR-257
Test |
Dosage (μL) |
Revertants per plate |
|||||||||||
TA-1353 |
TA-1357 |
TA-1358 |
TA-98 |
TA-100 |
D4* |
||||||||
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
||
Non-activation |
|||||||||||||
Solvent control |
- |
11 |
|
10 |
|
11 |
|
30 |
|
154 |
|
68 |
|
Positive control |
** |
236 |
|
901 |
|
408 |
|
721 |
|
530 |
|
>1000 |
|
Test compound |
0.001 |
15 |
|
5 |
|
11 |
|
20 |
|
191 |
|
48 |
|
0.01 |
8 |
|
13 |
|
8 |
|
24 |
|
203 |
|
55 |
|
|
0.1 |
25 |
|
6 |
|
5 |
|
19 |
|
212 |
|
54 |
|
|
1.0 |
19 |
|
9 |
|
5 |
|
21 |
|
198 |
|
44 |
|
|
5.0 |
12 |
|
10 |
|
5 |
|
20 |
|
210 |
|
39 |
|
|
Activation |
|||||||||||||
Solvent control |
- |
13 |
|
16 |
|
16 |
|
43 |
|
124 |
|
35 |
|
Positive control |
*** |
242 |
|
208 |
|
428 |
|
650 |
|
>1000 |
|
82 |
|
Test compound |
0.001 |
17 |
|
23 |
|
20 |
|
45 |
|
162 |
|
50 |
|
0.01 |
19 |
|
21 |
|
22 |
|
41 |
|
170 |
|
61 |
|
|
0.1 |
16 |
|
15 |
|
23 |
|
48 |
|
170 |
|
58 |
|
|
1.0 |
14 |
|
23 |
|
18 |
|
40 |
|
154 |
|
52 |
|
|
5.0 |
16 |
|
6 |
|
21 |
|
39 |
|
179 |
|
52 |
|
|
* TRY+ convertants per plate.
**
TA-1535 |
MNNG |
10 μg/plate |
TA-1537 |
QM |
10 μg/plate |
TA-1538 |
NF |
100 μg/plate |
TA-98 |
NF |
100 μg/plate |
TA-100 |
MNNG |
10 μg/plate |
D4 |
MNNG |
10 μg/plate |
***
TA-1535 |
ANTH |
100 μg/plate |
TA-1537 |
AMQ |
100 μg/plate |
TA-1538 |
AAF |
100 μg/plate |
TA-98 |
AAF |
100 μg/plate |
TA-100 |
ANTH |
100 μg/plate |
D4 |
DMNA |
100 micromoles/plate |
No raw data provided in study report.
No raw data provided in study report.
Table 1: Experiments without metabolic activation (S9 mix)
| 4 hr exposure | ||||
| Concentration [µg/mL medium] | CBPI | RI [%] | Number of binucleated cells scored | Number of micrnucleated cells per 1000 bionucleate cells |
| Highly purified water (vehicle controls) | ||||
| 0 | 1.35 | 100 | 2000 | 3.5 |
| Sodiu dihydrogen orthophosphate | ||||
| 125 | 1.36 | 103 | 2000 | 4.0 |
| 250 | 1.40 | 114 | 2000 | 3.0 |
| 500 | 1.48 | 139 | 2000 | 4.5 |
| 1000 | 1.38 | 109 | 2000 | 4.5 |
| 2000 | 1.36 | 205 | 2000 | 4.0 |
| Mitomycin C | ||||
| 0.2 | 1.38 | 109 | 2000 | 37.5 s |
| 24 -h exposure | ||||
| Concentration of test item [µg/mL medium | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells |
| Highly purified water (vehicle control) | ||||
| 0 | 1.30 | 100 | 2000 | 3.5 |
| Sodium dihydrogen orthophosphate | ||||
| 250 | 1.29 | 95 | 2000 | 3.0 |
| 500 | 1.31 | 104 | 2000 | 3.5 |
| 1000 | 1.26 | 87 | 2000 | 3.0 |
| 2000 | 1.26 | 87 | 2000 | 3.5 |
| Colchicine | ||||
| 0.02 | 1.21 | 70 | 2000 | 53.5 s |
CBPI = Cytokinesis block proliferation index
RI = Replicative Index
s. = significantly different from negative control (p ≤ 0.05)
Table 2: Experiment with metabolic activation (S9 mix)
| 4 -h exposure | ||||
| Concentration of test item [µg/mL medium] | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells |
| Highly purified water (vehicle contol) | ||||
| 0 | 1.31 | 100 | 2000 | 3.5 |
| Sodium dihydrogen orthophosphate | ||||
| 125 | 1.36 | 118 | 2000 | 3.5 |
| 250 | 1.29 | 94 | 2000 | 2.5 |
| 500 | 1.29 | 96 | 2000 | 4.5 |
| 1000 | 1.31 | 103 | 2000 | 3.5 |
| 2000 | 1.29 | 94 | 2000 | 3.5 |
| Cyclophospamide | ||||
| 20 | 1.29 | 93 | 2000 | 27.0 s |
CBPI = Cytokinesis block proliferation index
RI = Replicative Index
s. = significantly different from negative control (p ≤ 0.05)
Table 3: Experiment without metabolic activation (S9 mix) - 4 -hr exposure
| Culture number | Concentration [µg/mL medium] | mononucleate | Number of binucleate cells# | multinucleate | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells | Significance chi2 -test |
| Highly purified water (vehicle control) | |||||||||
| 1 | 0 | 345 | 144 | 11 | 1.33 | 100 | 1000 | 3 | - |
| 9 | 0 | 330 | 162 | 8 | 1.36 | 100 | 1000 | 4 | - |
| Sodium dihydrogen orthophosphate | |||||||||
| 6 | 125 | 340 | 155 | 5 | 1.33 | 100 | 1000 | 4 | n.s. |
| 14 | 125 | 320 | 169 | 11 | 1.38 | 106 | 1000 | 4 | n.s. |
| 5 | 250 | 342 | 146 | 12 | 1.34 | 103 | 1000 | 3 | n.s. |
| 13 | 250 | 296 | 181 | 23 | 1.45 | 125 | 1000 | 3 | n.s. |
| 4 | 500 | 246 | 237 | 17 | 1.54 | 164 | 1000 | 5 | n.s. |
| 12 | 500 | 306 | 183 | 11 | 1.41 | 114 | 1000 | 4 | n.s. |
| 3 | 1000 | 344 | 143 | 13 | 1.34 | 103 | 1000 | 4 | n.s. |
| 11 | 1000 | 310 | 177 | 13 | 1.41 | 114 | 1000 | 5 | n.s. |
| 2 | 2000 | 334 | 160 | 6 | 1.34 | 103 | 1000 | 4 | n.s. |
| 10 | 2000 | 320 | 172 | 8 | 1.38 | 106 | 1000 | 4 | n.s. |
| Mitmycin C | |||||||||
| 7 | 0.2 | 309 | 183 | 8 | 1.40 | 121 | 1000 | 36 | s. |
| 15 | 0.2 | 326 | 171 | 3 | 135 | 97 | 1000 | 39 | s. |
n.s. = not significantly different from negative control (p ≤ 0.05)
s. = significantly different from negative control (p ≤ 0.05)
CBPI = Cytokinesis block proliferation index
RI = Replicative test
Table 4: Experiment without metabolic activation (S9 mix) - 24hr exposure
| Culture number | Concentration [µg/mL medium] | mononucleate | Number of binucleate cells# | multinucleate | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells | Significane chi2-test |
| Highly ourified water (vehicle control) | |||||||||
| 1 | 0 | 354 | 134 | 12 | 1.32 | 100 | 1000 | 4 | - |
| 9 | 0 | 378 | 106 | 16 | 1.28 | 100 | 1000 | 3 | - |
| Sodium dihydrogen orthophosphate | |||||||||
| 5 | 250 | 366 | 105 | 29 | 1.33 | 103 | 1000 | 4 | n.s. |
| 13 | 250 | 379 | 120 | 1 | 1.24 | 86 | 1000 | 2 | n.s. |
| 4 | 500 | 363 | 122 | 15 | 1.30 | 94 | 1000 | 5 | n.s. |
| 12 | 500 | 356 | 130 | 14 | 1.32 | 114 | 1000 | 2 | n.s. |
| 3 | 1000 | 377 | 110 | 13 | 1.27 | 84 | 1000 | 3 | n.s. |
| 11 | 1000 | 382 | 112 | 6 | 1.25 | 89 | 1000 | 3 | n.s. |
| 2 | 2000 | 370 | 119 | 11 | 1.28 | 88 | 1000 | 4 | n.s. |
| 10 | 2000 | 389 | 103 | 8 | 1.24 | 86 | 1000 | 3 | n.s. |
| Colchicine | |||||||||
| 7 | 0.02 | 403 | 86 | 11 | 1.22 | 69 | 1000 | 61 | s. |
| 15 | 0.02 | 408 | 85 | 7 | 1.20 | 71 | 1000 | 46 | s. |
n.s = not significantly different from negative control (p ≤ 0.05)
s. = significantly different from negative control (p ≤ 0.05)
CBPI = Cytokinesis block proliferation index
RI = Replicative Index
Table 5: Experiment with metabolic activation (+S9 mix) -4hr exposure
| Culture number | Concentration [µg/mL medium] | mononucleate | Number of binucleate cells# | multinucleate | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells | Significance chi2-test |
| Highly purified water (vehicle control) | |||||||||
| 1 | 0 | 366 | 128 | 6 | 1.28 | 100 | 1000 | 4 | - |
| 9 | 0 | 343 | 143 | 14 | 1.34 | 100 | 1000 | 3 | - |
| Sodium dihydrogen orthphosphate | |||||||||
| 6 | 125 | 320 | 165 | 15 | 1.39 | 139 | 1000 | 4 | n.s. |
| 14 | 125 | 345 | 146 | 9 | 1.33 | 97 | 1000 | 3 | n.s. |
| 5 | 250 | 367 | 127 | 6 | 1.28 | 100 | 1000 | 2 | n.s. |
| 13 | 250 | 362 | 127 | 11 | 1.30 | 88 | 1000 | 3 | n.s |
| 4 | 500 | 344 | 144 | 12 | 1.34 | 121 | 1000 | 4 | n.s. |
| 12 | 500 | 288 | 103 | 9 | 1.24 | 71 | 1000 | 5 | n.s. |
| 3 | 1000 | 325 | 163 | 12 | 1.37 | 132 | 1000 | 3 | n.s. |
| 11 | 1000 | 380 | 116 | 4 | 1.25 | 74 | 1000 | 4 | n.s. |
| 2 | 2000 | 378 | 106 | 16 | 1.28 | 100 | 1000 | 2 | n.s. |
| 10 | 2000 | 361 | 130 | 9 | 1.30 | 88 | 1000 | 5 | n.s. |
| Cyclophosphamide | |||||||||
| 7 | 20 | 367 | 127 | 6 | 1.28 | 100 | 1000 | 26 | s. |
| 15 | 20 | 362 | 132 | 6 | 1.29 | 85 | 1000 | 28 | s. |
n.s. = not significantly different from negative control (p ≤ 0.05)
s. = significantly different from negative control (p ≤ 0.05)
CBPI = Cytokinesis block proliferation index
RI replicative index
Table 1: Experiments without metabolic activation (S9 mix)
| 4 hr exposure | ||||
| Concentration [µg/mL medium] | CBPI | RI [%] | Number of binucleated cells scored | Number of micrnucleated cells per 1000 bionucleate cells |
| Highly purified water (vehicle controls) | ||||
| 0 | 1.35 | 100 | 2000 | 3.5 |
| Sodiu dihydrogen orthophosphate | ||||
| 125 | 1.36 | 103 | 2000 | 4.0 |
| 250 | 1.40 | 114 | 2000 | 3.0 |
| 500 | 1.48 | 139 | 2000 | 4.5 |
| 1000 | 1.38 | 109 | 2000 | 4.5 |
| 2000 | 1.36 | 205 | 2000 | 4.0 |
| Mitomycin C | ||||
| 0.2 | 1.38 | 109 | 2000 | 37.5 s |
| 24 -h exposure | ||||
| Concentration of test item [µg/mL medium | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells |
| Highly purified water (vehicle control) | ||||
| 0 | 1.30 | 100 | 2000 | 3.5 |
| Sodium dihydrogen orthophosphate | ||||
| 250 | 1.29 | 95 | 2000 | 3.0 |
| 500 | 1.31 | 104 | 2000 | 3.5 |
| 1000 | 1.26 | 87 | 2000 | 3.0 |
| 2000 | 1.26 | 87 | 2000 | 3.5 |
| Colchicine | ||||
| 0.02 | 1.21 | 70 | 2000 | 53.5 s |
CBPI = Cytokinesis block proliferation index
RI = Replicative Index
s. = significantly different from negative control (p ≤ 0.05)
Table 2: Experiment with metabolic activation (S9 mix)
| 4 -h exposure | ||||
| Concentration of test item [µg/mL medium] | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells |
| Highly purified water (vehicle contol) | ||||
| 0 | 1.31 | 100 | 2000 | 3.5 |
| Sodium dihydrogen orthophosphate | ||||
| 125 | 1.36 | 118 | 2000 | 3.5 |
| 250 | 1.29 | 94 | 2000 | 2.5 |
| 500 | 1.29 | 96 | 2000 | 4.5 |
| 1000 | 1.31 | 103 | 2000 | 3.5 |
| 2000 | 1.29 | 94 | 2000 | 3.5 |
| Cyclophospamide | ||||
| 20 | 1.29 | 93 | 2000 | 27.0 s |
CBPI = Cytokinesis block proliferation index
RI = Replicative Index
s. = significantly different from negative control (p ≤ 0.05)
Table 3: Experiment without metabolic activation (S9 mix) - 4 -hr exposure
| Culture number | Concentration [µg/mL medium] | mononucleate | Number of binucleate cells# | multinucleate | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells | Significance chi2 -test |
| Highly purified water (vehicle control) | |||||||||
| 1 | 0 | 345 | 144 | 11 | 1.33 | 100 | 1000 | 3 | - |
| 9 | 0 | 330 | 162 | 8 | 1.36 | 100 | 1000 | 4 | - |
| Sodium dihydrogen orthophosphate | |||||||||
| 6 | 125 | 340 | 155 | 5 | 1.33 | 100 | 1000 | 4 | n.s. |
| 14 | 125 | 320 | 169 | 11 | 1.38 | 106 | 1000 | 4 | n.s. |
| 5 | 250 | 342 | 146 | 12 | 1.34 | 103 | 1000 | 3 | n.s. |
| 13 | 250 | 296 | 181 | 23 | 1.45 | 125 | 1000 | 3 | n.s. |
| 4 | 500 | 246 | 237 | 17 | 1.54 | 164 | 1000 | 5 | n.s. |
| 12 | 500 | 306 | 183 | 11 | 1.41 | 114 | 1000 | 4 | n.s. |
| 3 | 1000 | 344 | 143 | 13 | 1.34 | 103 | 1000 | 4 | n.s. |
| 11 | 1000 | 310 | 177 | 13 | 1.41 | 114 | 1000 | 5 | n.s. |
| 2 | 2000 | 334 | 160 | 6 | 1.34 | 103 | 1000 | 4 | n.s. |
| 10 | 2000 | 320 | 172 | 8 | 1.38 | 106 | 1000 | 4 | n.s. |
| Mitmycin C | |||||||||
| 7 | 0.2 | 309 | 183 | 8 | 1.40 | 121 | 1000 | 36 | s. |
| 15 | 0.2 | 326 | 171 | 3 | 135 | 97 | 1000 | 39 | s. |
n.s. = not significantly different from negative control (p ≤ 0.05)
s. = significantly different from negative control (p ≤ 0.05)
CBPI = Cytokinesis block proliferation index
RI = Replicative test
Table 4: Experiment without metabolic activation (S9 mix) - 24hr exposure
| Culture number | Concentration [µg/mL medium] | mononucleate | Number of binucleate cells# | multinucleate | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells | Significane chi2-test |
| Highly ourified water (vehicle control) | |||||||||
| 1 | 0 | 354 | 134 | 12 | 1.32 | 100 | 1000 | 4 | - |
| 9 | 0 | 378 | 106 | 16 | 1.28 | 100 | 1000 | 3 | - |
| Sodium dihydrogen orthophosphate | |||||||||
| 5 | 250 | 366 | 105 | 29 | 1.33 | 103 | 1000 | 4 | n.s. |
| 13 | 250 | 379 | 120 | 1 | 1.24 | 86 | 1000 | 2 | n.s. |
| 4 | 500 | 363 | 122 | 15 | 1.30 | 94 | 1000 | 5 | n.s. |
| 12 | 500 | 356 | 130 | 14 | 1.32 | 114 | 1000 | 2 | n.s. |
| 3 | 1000 | 377 | 110 | 13 | 1.27 | 84 | 1000 | 3 | n.s. |
| 11 | 1000 | 382 | 112 | 6 | 1.25 | 89 | 1000 | 3 | n.s. |
| 2 | 2000 | 370 | 119 | 11 | 1.28 | 88 | 1000 | 4 | n.s. |
| 10 | 2000 | 389 | 103 | 8 | 1.24 | 86 | 1000 | 3 | n.s. |
| Colchicine | |||||||||
| 7 | 0.02 | 403 | 86 | 11 | 1.22 | 69 | 1000 | 61 | s. |
| 15 | 0.02 | 408 | 85 | 7 | 1.20 | 71 | 1000 | 46 | s. |
n.s = not significantly different from negative control (p ≤ 0.05)
s. = significantly different from negative control (p ≤ 0.05)
CBPI = Cytokinesis block proliferation index
RI = Replicative Index
Table 5: Experiment with metabolic activation (+S9 mix) -4hr exposure
| Culture number | Concentration [µg/mL medium] | mononucleate | Number of binucleate cells# | multinucleate | CBPI | RI [%] | Number of binucleate cells scored | Number of micronucleated cells per 1000 binucleate cells | Significance chi2-test |
| Highly purified water (vehicle control) | |||||||||
| 1 | 0 | 366 | 128 | 6 | 1.28 | 100 | 1000 | 4 | - |
| 9 | 0 | 343 | 143 | 14 | 1.34 | 100 | 1000 | 3 | - |
| Sodium dihydrogen orthphosphate | |||||||||
| 6 | 125 | 320 | 165 | 15 | 1.39 | 139 | 1000 | 4 | n.s. |
| 14 | 125 | 345 | 146 | 9 | 1.33 | 97 | 1000 | 3 | n.s. |
| 5 | 250 | 367 | 127 | 6 | 1.28 | 100 | 1000 | 2 | n.s. |
| 13 | 250 | 362 | 127 | 11 | 1.30 | 88 | 1000 | 3 | n.s |
| 4 | 500 | 344 | 144 | 12 | 1.34 | 121 | 1000 | 4 | n.s. |
| 12 | 500 | 288 | 103 | 9 | 1.24 | 71 | 1000 | 5 | n.s. |
| 3 | 1000 | 325 | 163 | 12 | 1.37 | 132 | 1000 | 3 | n.s. |
| 11 | 1000 | 380 | 116 | 4 | 1.25 | 74 | 1000 | 4 | n.s. |
| 2 | 2000 | 378 | 106 | 16 | 1.28 | 100 | 1000 | 2 | n.s. |
| 10 | 2000 | 361 | 130 | 9 | 1.30 | 88 | 1000 | 5 | n.s. |
| Cyclophosphamide | |||||||||
| 7 | 20 | 367 | 127 | 6 | 1.28 | 100 | 1000 | 26 | s. |
| 15 | 20 | 362 | 132 | 6 | 1.29 | 85 | 1000 | 28 | s. |
n.s. = not significantly different from negative control (p ≤ 0.05)
s. = significantly different from negative control (p ≤ 0.05)
CBPI = Cytokinesis block proliferation index
RI replicative index
Please see Attached Tables
Due to the nature and quantity of the tables it was not possible to insert them in this section.
Text table 7-1 pH values and osmolality
Concentration of |
pH value |
Osmolality |
Sodium dihydrogenorthophosphate |
|
[mOsmol/kg] |
[µg/mL medium] |
|
|
Medium |
7.59 |
290.0 |
Negative control |
7.60 |
290.0 |
10 |
7.62 |
285.0 |
31.6 |
7.65 |
285.0 |
100 |
7.53 |
285.0 |
316 |
7.67 |
285.0 |
1000 |
6.96 |
290.0 |
2000 |
6.68 |
310.0 |
Please see Attached Tables
Due to the nature and quantity of the tables it was not possible to insert them in this section.
Text table 7-1 pH values and osmolality
Concentration of |
pH value |
Osmolality |
Sodium dihydrogenorthophosphate |
|
[mOsmol/kg] |
[µg/mL medium] |
|
|
Medium |
7.59 |
290.0 |
Negative control |
7.60 |
290.0 |
10 |
7.62 |
285.0 |
31.6 |
7.65 |
285.0 |
100 |
7.53 |
285.0 |
316 |
7.67 |
285.0 |
1000 |
6.96 |
290.0 |
2000 |
6.68 |
310.0 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
No classification for in vitro genetic toxicity is proposed. This is based on a weight of evidence approach using all relevant data on sodium acid pyrophosphate and its analogues. No further testing is considered necessary for the following reasons:
Sodium and potassium phosphates are routinely used in the nutrient broths that support cell cultures in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, disodium dihydrogenpyrophosphate will hydrolyse to sodium orthophosphates which are also found in the metabolic activation mixture (e.g. S9-mix) which is used in a chromosomal aberration test to determine whether a test chemical can be metabolized within the body to produce a compound that may be genotoxic. The constant exposure of bacteria to these materials suggests that they pose no inherent risk of genotoxicity.
As such and in accordance with Regulation (EC) No.1272/2008 (EU CLP) no classification is proposed.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
