Disodium dihydrogenpyrophosphate

Endpoint summary

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

Reference

Reference 1

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. 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'.

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)	-	-	>10^3	>10^3
Positive control – QM (0.25 mg)	-	-			>10^2	>10^2
Positive control – NF (0.25 mg)	-	-					>10^2	>10^2
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	>10^3	>10^3
Positive control – AAF		Liver			44	43	>10^2	>10^2
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	>10^2	>10^2
Positive control – AAF (1.25 mg)		Liver			41	30	>10^2	>10^2
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	>10^2	>10^2
Positive control – AAF (1.25 mg)		Liver			32	46	>10^2	>10^2
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

 

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 data is submitted to provide a weight of evidence to support the conclusion that disodium dihydrogenpyrophosphate is unlikely to produce a positive genotoxic response.

Reference 2

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

Remarks:

.

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. 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'.

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 ± 213^1
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

Conclusions:

The authors conclude that sodium pyrophosphate is non-mutagenic under the conditions of this study.
This data is submitted to provide a weight of evidence to support the conclusion that disodium dihydrogenpyrophosphate is unlikely to produce a positive genotoxic response.

Reference 3

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

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'.

Table 1: Results as presented in study:

Additive	Max dose (mg/plate)	Result
Disodium dihydrogen phosphate	10.0	Negative

Conclusions:

Disodium dihydrogen phosphate is considered to be non-mutagenic in strains of S.typhimurium, under the conditions of this assay.

This data is submitted to provide a weight of evidence to support the conclusion that disodium dihydrogenpyrophosphate is unlikely to produce a positive genotoxic response.

Reference 4

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. 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:

Stduy 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'.

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

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 data is submitted to provide a weight of evidence to support the conclusion that disodium dihydrogenpyrophosphate is unlikely to produce a positive genotoxic response.

Reference 5

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. 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 and therefore GLP compliance not specified.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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.