Tetrapotassium pyrophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A number of studies are available to assess the genotoxic potential of tetrapotassium pyrophosphate and the analogous substances tetrasodium pyrophosphate and disodium dihydrogenpyrophosphate. Sodium and potassium pyrophosphates are considered to be non-genotoxic in all studies performed and therefore no further testing is required.

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

Remarks:

Although study design is comparable to OECD 471, the data does not meet important criteria of today’s standard methods. OECD 471 states that in order to detect cross-linking mutagens it may be preferable to include S. typhimurium strain TA 102 or a DNA-proficient strain of E.coli, neither of which are considered in the study and only 3 strains of S. typhimurium were tested. In addition a non-standard assay was conducted on a strain of S.cerivisiae.

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'.

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:

Interpretation of results:
negative with and without metabolic activation

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 tetrapotassium pyrophosphate 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:

Documentation insufficient for assessment: Paper is written in Japanese with abstract and tabulated data only in English.

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:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Used in tester strain TA 97 without S9 Migrated to IUCLID6: 50 µg

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Used in tester strain TA 102 without S9 Migrated to IUCLID6: 0.5 µg

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (5 µg)

Remarks:

Used in tester strain TA 97 and TA 102 with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- 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:

not specified

Additional information on results:

No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Results of a mutation test with positive controls

Substance	Solvent	Dose (µg/plate)	No. of revertant / 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 a mutagenicity study conducted on potassium pyrophosphate

Substance	Solvent	Dose (mg/plate)	No. of revertant
			TA 97		TA 102
			-S9	+S9	-S9	+S9
Potassium pyrophosphate	DW	10	133	146	269	244
		5	125	191	299	277
		1	82	170	287	425
		0.5	137	187	271	468
		0.1	131	187	296	485
		0	114	183	292	478

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The authors conclude that potassium 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.
This data is submitted to provide a weight of evidence to support the conclusion that tetrapotassium pyrophosphate 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:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Documentation insufficient for assessment: Paper is written in Japanese with abstract and tabulated data only in English.

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'.

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:

Interpretation of results:
negative with metabolic activation
ambiguous without metabolic activation

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 tetrapotassium pyrophosphate 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

Remarks:

Significant methodological deficiencies: Study design is comparable to OECD 471, however, the following deficiencies in the study design and reporting of results exist. No positive control data are determined, individual data scores relating to strain and dose level are not included in the study report and only one dose level is reported.

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'.

Table 1: Results as presented in study:

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

Conclusions:

Interpretation of results:
negative

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 tetrapotassium pyrophosphate 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:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Although study design is comparable to OECD 471, the data does not meet important criteria of today’s standard methods. OECD 471 states that in order to detect cross-linking mutagens it may be preferable to include S. typhimurium strain TA 102 or a DNA-proficient strain of E.coli, neither of which are considered in the study. In addition, the substance identity is not clearly defined in terms of purity, composition or chemical identity.

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:

Interpretation of results:
negative With and without metabolic activation

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 tetrapotassium pyrophosphate is unlikely to produce a positive genotoxic response.

Reference 6

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 details on GLP compliance

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:

Interpretation of results:
negative

Tetrasodium pyrophosphate was found to be non-genotoxic in an AMES test both with and without metabolic activation.

Reference 7

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

Remarks:

Although study design is comparable to OECD 471, the data does not meet important criteria of today’s standard methods. OECD 471 states that in order to detect cross-linking mutagens it may be preferable to include S. typhimurium strain TA 102 or a DNA-proficient strain of E.coli, neither of which are considered in the study and only 3 strains of S. typhimurium were tested.

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'.

No raw data provided in study report.

Conclusions:

Interpretation of results:
negative with metabolic activation

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 data is submitted to provide a weight of evidence to support the conclusion that the analogous substance tetrasodium pyrophosphate is unlikely to produce a positive genotoxic response.
This data is submitted to provide a weight of evidence to support the conclusion that tetrapotassium pyrophosphate is unlikely to produce a positive genotoxic response.

Reference 8

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

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 chi^2 -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 chi^2-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 chi^2-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

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.

Reference 9

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.

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

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 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 tetrapotassium pyrophosphate and analogous substances and scientific justification for no further testing 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, tetrapotassium pyrophosphate will hydrolyse to potassium 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.

 

In addition the Na+, K+ and PO₄^3-ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems. As such and in accordance with Regulation (EC) No.1272/2008 (EU CLP) no classification is proposed.