Trisodium orthophosphate

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 is available for the Annex VIII Section 8.4.2 in vitro cytogenicity and Annex VIII Section 8.4.3 in vitro gene mutation study in mammalian cells endpoints.

A range of supporting studies are available for all endpoints. These include studies classed as Klimisch Reliability 1 on phosphoric acid.

None of the studies suggest the substance ot analogous substances are mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

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

Principles of method if other than guideline:

In addition to the study in S. typhimurium, the same assay was also performed on Saccharomyces cerevisiae, strain: D4.

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 liver, lung and testes of ICR random bred male mice, Sprague-Dawley adult male rats and Macaca mulatta adult male primates

Test concentrations with justification for top dose:

5.0%

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Used in a non-activation assay with tester strain TA 1535 Migrated to IUCLID6: 10 µL/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: quinacrine mustard ( 20 µg/plate)

Remarks:

Used in a non-activation assay with tester strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Used in a non-activation assay with tester strain TA 1538 Migrated to IUCLID6: 100 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: dimethylnitrosamine (50 µM/plate)

Remarks:

Used in an activation assay with tester strain TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (100 µg/plate)

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble under treatment conditions

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test date for toxicity determinations: April 11, 1975
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): 10.0, 1.0, 0.1, 0.01, 0.001.
-Concentrations of the test compound used in mutagenicity tests:

BACTERIA:
- 1/4 50% survival: 2.5%
-1/2 50% survival: 5.0%
-50% survival: 10.0%
-Plate tests: 5.0%

YEAST:
- 1/4 50% survival: 2.5%
-1/2 50% survival: 5.0%
-50% survival: 10.0%
-Plate tests: not applicable

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
Solvent control	-	-	3	4	22	33	7	11
Positive control^a	-	-	>10^3	>10^3	138	133	146	138
Test compound	-	-	8	3	21	17	9	10
2. Activation
Negative control	---	-	6	4	27	23	7	14
Solvent control		-	10	12	36	43	16	18
Reaction mixture control		-	9	7	36	39	19	23
Positive control^b	Mouse	Liver	>10^3	>10^3	96	93	333	317
Positive control		Lung	9	7	33	33	21	26
Positive control		Testes	7	2	37	32	18	11
Positive control	Rat	Liver	>10^3	>10^3	84	80	314	317
Positive control		Lung	11	6	32	35	15	18
Positive control		Testes	6	5	24	43	20	9
Positive control	Monkey	Liver	>10^3	>10^3	97	93	111	155
Positive control		Lung	8	5	32	38	13	15
Positive control		Testes	8	2	28	33	12	8
Test compound	Mouse	Liver	3	4	45	46	13	7
Test compound		Lung	4	7	30	20	8	12
Test compound		Testes	9	9	23	26	17	10
Test compound	Rat	Liver	3	4	46	46	13	8
Test compound		Lung	4	9	30	22	12	14
Test compound		Testes	9	9	28	27	13	13
Test compound	Monkey	Liver	3	5	46	49	13	9
Test compound		Lung	4	7	23	18	10	12
Test compound		Testes	9	9	19	22	13	9

 

^aTA 1353:. TA 1537: QM, TA 1538: NF

^bTA 1353: DMNA, TA 1537: AAF, TA 1538: AAF

 

 

Table 3: compound frequency summary report – suspension tests

 

Test	Test strain
	TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Nonactivation: solvent control	12.94	5.32	1.24	1.89	1.48
Nonactivation: positive control	771.32	1764.20	992.93	129.05	155.87
Nonactivation: dose 1	3.57	6.67	2.04	1.54	1.69
Nonactivation: dose 2	3.03	6.77	1.67	0.76	2.14

 

 

Table 4: Compound frequency summary report – suspension tests: with mouse-derived metabolic preparations

 

Test	ORG	Test strain
		TA 1535	TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	3.23		20.45	6.88	2.25	1.59
Activation	Tissue control	7.69		35.90	6.31	3.25	1.59
Activation	Solvent control	3.73	7.45	12.22	2.93	0.96	0.17
Activation	PLI	5496.88		45.07	15.26	7.59	6.16
Activation	PLU	4.79		20.50	4.48	2.42	2.60
Activation	PTE	9.15		28.79	2.33	3.81	2.06
Activation	Liver activation fraction – dose 1	6.29		37.82	7.38	2.27	1.98
Activation	Liver activation fraction – dose 2	22.34	6.06	11.39	5.85	1.61	3.03
Activation	Lung activation fraction – dose 1	5.50		5.98	3.75	1.80	2.20
Activation	Lung activation fraction – dose 2	3.94		11.62	3.91	1.86	1.86
Activation	Testes activation fraction – dose 1	2.86		22.38	4.07	3.26	2.34
Activation	Testes activation fraction – dose 2	4.18		23.86	3.77	2.08	2.08

 

 

Table 5: Compound frequency summary report – suspension tests: with rat-derived metabolic preparations

 

 

Test	ORG	Test strain
		TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	4.48	10.86	4.75	2.65	2.37
Activation	Tissue control	2.48	10.31	6.57	3.90	3.90
Activation	Solvent control	5.52	8.87	9.05	2.94	3.03
Activation	PLI	333.15	17.19	20.33	7.37	9.16
Activation	PLU	6.11	14.09	8.76	3.32	19.54
Activation	PTE	8.60	11.40	6.77	3.84	4.23
Activation	Liver activation fraction – dose 1	2.17	9.77	11.53	1.98	1.72
Activation	Liver activation fraction – dose 2	2.95	8.47	10.87	3.06	2.45
Activation	Lung activation fraction – dose 1	5.65	10.54	8.94	2.14	2.05
Activation	Lung activation fraction – dose 2	5.49	11.50	8.49	2.65	2.47
Activation	Testes activation fraction – dose 1	3.91	11.05	14.46	3.59	3.04
Activation	Testes activation fraction – dose 2	5.26	13.72	4.64	3.58	2.08

 

 

Table 6: Compound frequency summary report – suspension tests: with monkey-derived metabolic preparations

 

 

Test	ORG	Test strain
		TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	4.50	6.36	10.43	1.82	1.82
Activation	Tissue control	5.62	3.92	5.80	2.59	2.73
Activation	Solvent control	7.72	5.63	2.70	2.16	1.80
Activation	PLI	1194.98	10.56	54.55	6.38	3.75
Activation	PLU	5.81	5.60	5.22	2.40	2.64
Activation	PTE	3.99	8.26	6.61	5.41	2.30
Activation	Liver activation fraction – dose 1	6.58	5.54	2.94	1.73	1.51
Activation	Liver activation fraction – dose 2	10.47	7.69	6.43	2.90	2.53
Activation	Lung activation fraction – dose 1	4.44	6.97	7.67	3.42	2.65
Activation	Lung activation fraction – dose 2	5.48	6.09	2.71	2.37	2.37
Activation	Testes activation fraction – dose 1	3.76	5.93	5.88	3.22	1.33
Activation	Testes activation fraction – dose 2	4.18	7.16	4.53	2.42	2.42

Conclusions:

Monopotassium phosphate granular food grade is considered to be non-mutagenic under the conditions of this study.


This study is considered to be sufficient to fulfil this endpoint as part of a weight of evidence and no further testing will be required for the following reasons:

As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting 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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA 1353, TA 1357, TA 1358

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble under treatment conditions

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test date for toxicity determinations: April 11, 1975
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): 10.0, 1.0, 0.1, 0.01, 0.001.
-Concentrations of the test compound used in mutagenicity tests:

BACTERIA:
- 1/4 50% survival: 2.5%
-1/2 50% survival: 5.0%
-50% survival: 10.0%
-Plate tests: 5.0%

YEAST:
- 1/4 50% survival: 2.5%
-1/2 50% survival: 5.0%
-50% survival: 10.0%
-Plate tests: not applicable

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
Solvent control	-	-	3	4	22	33	7	11
Positive control^a	-	-	>10^3	>10^3	138	133	146	138
Test compound	-	-	8	3	21	17	9	10
2. Activation
Negative control	---	-	6	4	27	23	7	14
Solvent control		-	10	12	36	43	16	18
Reaction mixture control		-	9	7	36	39	19	23
Positive control^b	Mouse	Liver	>10^3	>10^3	96	93	333	317
Positive control		Lung	9	7	33	33	21	26
Positive control		Testes	7	2	37	32	18	11
Positive control	Rat	Liver	>10^3	>10^3	84	80	314	317
Positive control		Lung	11	6	32	35	15	18
Positive control		Testes	6	5	24	43	20	9
Positive control	Monkey	Liver	>10^3	>10^3	97	93	111	155
Positive control		Lung	8	5	32	38	13	15
Positive control		Testes	8	2	28	33	12	8
Test compound	Mouse	Liver	3	4	45	46	13	7
Test compound		Lung	4	7	30	20	8	12
Test compound		Testes	9	9	23	26	17	10
Test compound	Rat	Liver	3	4	46	46	13	8
Test compound		Lung	4	9	30	22	12	14
Test compound		Testes	9	9	28	27	13	13
Test compound	Monkey	Liver	3	5	46	49	13	9
Test compound		Lung	4	7	23	18	10	12
Test compound		Testes	9	9	19	22	13	9

 

^aTA 1353:. TA 1537: QM, TA 1538: NF

^bTA 1353: DMNA, TA 1537: AAF, TA 1538: AAF

 

 

Table 3: compound frequency summary report – suspension tests

 

Test	Test strain
	TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Nonactivation: solvent control	12.94	5.32	1.24	1.89	1.48
Nonactivation: positive control	771.32	1764.20	992.93	129.05	155.87
Nonactivation: dose 1	3.57	6.67	2.04	1.54	1.69
Nonactivation: dose 2	3.03	6.77	1.67	0.76	2.14

 

 

Table 4: Compound frequency summary report – suspension tests: with mouse-derived metabolic preparations

 

Test	ORG	Test strain
		TA 1535	TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	3.23		20.45	6.88	2.25	1.59
Activation	Tissue control	7.69		35.90	6.31	3.25	1.59
Activation	Solvent control	3.73	7.45	12.22	2.93	0.96	0.17
Activation	PLI	5496.88		45.07	15.26	7.59	6.16
Activation	PLU	4.79		20.50	4.48	2.42	2.60
Activation	PTE	9.15		28.79	2.33	3.81	2.06
Activation	Liver activation fraction – dose 1	6.29		37.82	7.38	2.27	1.98
Activation	Liver activation fraction – dose 2	22.34	6.06	11.39	5.85	1.61	3.03
Activation	Lung activation fraction – dose 1	5.50		5.98	3.75	1.80	2.20
Activation	Lung activation fraction – dose 2	3.94		11.62	3.91	1.86	1.86
Activation	Testes activation fraction – dose 1	2.86		22.38	4.07	3.26	2.34
Activation	Testes activation fraction – dose 2	4.18		23.86	3.77	2.08	2.08

 

 

Table 5: Compound frequency summary report – suspension tests: with rat-derived metabolic preparations

 

 

Test	ORG	Test strain
		TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	4.48	10.86	4.75	2.65	2.37
Activation	Tissue control	2.48	10.31	6.57	3.90	3.90
Activation	Solvent control	5.52	8.87	9.05	2.94	3.03
Activation	PLI	333.15	17.19	20.33	7.37	9.16
Activation	PLU	6.11	14.09	8.76	3.32	19.54
Activation	PTE	8.60	11.40	6.77	3.84	4.23
Activation	Liver activation fraction – dose 1	2.17	9.77	11.53	1.98	1.72
Activation	Liver activation fraction – dose 2	2.95	8.47	10.87	3.06	2.45
Activation	Lung activation fraction – dose 1	5.65	10.54	8.94	2.14	2.05
Activation	Lung activation fraction – dose 2	5.49	11.50	8.49	2.65	2.47
Activation	Testes activation fraction – dose 1	3.91	11.05	14.46	3.59	3.04
Activation	Testes activation fraction – dose 2	5.26	13.72	4.64	3.58	2.08

 

 

Table 6: Compound frequency summary report – suspension tests: with monkey-derived metabolic preparations

 

 

Test	ORG	Test strain
		TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	4.50	6.36	10.43	1.82	1.82
Activation	Tissue control	5.62	3.92	5.80	2.59	2.73
Activation	Solvent control	7.72	5.63	2.70	2.16	1.80
Activation	PLI	1194.98	10.56	54.55	6.38	3.75
Activation	PLU	5.81	5.60	5.22	2.40	2.64
Activation	PTE	3.99	8.26	6.61	5.41	2.30
Activation	Liver activation fraction – dose 1	6.58	5.54	2.94	1.73	1.51
Activation	Liver activation fraction – dose 2	10.47	7.69	6.43	2.90	2.53
Activation	Lung activation fraction – dose 1	4.44	6.97	7.67	3.42	2.65
Activation	Lung activation fraction – dose 2	5.48	6.09	2.71	2.37	2.37
Activation	Testes activation fraction – dose 1	3.76	5.93	5.88	3.22	1.33
Activation	Testes activation fraction – dose 2	4.18	7.16	4.53	2.42	2.42

Conclusions:

Trisodium phosphate is estimated to be non-mutagenic under the conditions of this study.

Executive summary:

Trisodium phosphate was estimated to be non-mutagenic as found in the source study performed with monopotassium phosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium phosphate and monopotassium phosphate are unlikely to lead to differences in the genetic toxicity that are higher than the typical experimental error of the test method

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

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

Principles of method if other than guideline:

In addition to the study in S. typhimurium, the same assay was also performed on Saccharomyces cerevisiae, strain: D4.

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:

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 liver, lung and testes of ICR random bred male mice, Sprague-Dawley adult male rats and Macaca mulatta adult male primates

Test concentrations with justification for top dose:

1.25%

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Used in a non-activation assay with tester strain TA 1535 and S. cerevisiae D4 Migrated to IUCLID6: 10 µL/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: quinacrine mustard ( 20 µg/plate)

Remarks:

Used in a non-activation assay with tester strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Used in a non-activation assay with tester strain TA 1538 Migrated to IUCLID6: 100 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: dimethylnitrosamine (50 µM/plate)

Remarks:

Used in an activation assay with tester strain TA 1535 and S. cerevisiae D4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (100 µg/plate)

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: soluble under treatment conditions

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test date for toxicity determinations: March 25, 1975
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): 10.0, 1.0, 0.1, 0.01, 0.001.
-Concentrations of the test compound used in mutagenicity tests:

BACTERIA:
- 1/4 50% survival: 0.625%
-1/2 50% survival: 1.250%
-50% survival: 2.500%
-Plate tests: 1.250%

YEAST:
- 1/4 50% survival: 2.5%
-1/2 50% survival: 5.0%
-50% survival: 10.0%
-Plate tests: not applicable

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
Solvent control	-	-	40	41	11	13	27	33
Positive control^a	-	-	>10^3	>10^3	183	113	98	129
Test compound	-	-	44	46	8	14	15	22
2. Activation
Negative control	---	-	8	12	7	7	6	18
Solvent control		-	13	4	15	16	23	21
Reaction mixture control		-	7	10	8	8	10	18
Positive control^b	Mouse	Liver	>10^3	>10^3	41	43	307	420
Positive control		Lung	11	13	5	12	72	30
Positive control		Testes	9	11	22	10	19	22
Positive control	Rat	Liver	>10^3	>10^3	41	45	327	340
Positive control		Lung	12	9	7	7	26	29
Positive control		Testes	9	11	22	10	19	22
Positive control	Monkey	Liver	390	329	44	41	363	310
Positive control		Lung	11	9	7	10	21	24
Positive control		Testes	9	12	16	6	17	12
Test compound	Mouse	Liver	19	16	10	22	24	20
Test compound		Lung	23	20	7	5	17	21
Test compound		Testes	17	24	8	9	20	31
Test compound	Rat	Liver	16	14	10	14	23	15
Test compound		Lung	21	18	6	9	20	18
Test compound		Testes	17	22	7	10	21	29
Test compound	Monkey	Liver	19	15	15	24	20	14
Test compound		Lung	23	16	8	8	18	21
Test compound		Testes	21	20	7	8	19	29

 

^aTA 1353:. TA 1537: QM, TA 1538: NF

^bTA 1353: DMNA, TA 1537: AAF, TA 1538: AAF

 

 

Table 3: compound frequency summary report – suspension tests

 

Test	Test strain
	TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Nonactivation: solvent control	7.58	23.63	15.00	1.07	0.93
Nonactivation: positive control	376.09	688.06	666.27	2.08	117.36
Nonactivation: dose 1	13.02	14.14	10.00	1.81	1,27
Nonactivation: dose 2	11.71	14.62	8.08	2.74	1.46

 

 

Table 4: Compound frequency summary report – suspension tests: with mouse-derived metabolic preparations. The LI1, LI2 and LU2 dose levels were repeated with TA 1537 because of increased mutant frequencies. The repeat tests were negative.

 

Test	ORG	Test strain
		TA 1535	TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)	TA 1537
Activation	Negative chemical control	18.11	4.61	10.20		2.12	1.52
Activation	Tissue control	14.08	9.87	14.37		3.14	5.10
Activation	Solvent control	7.98	4.84	10.54	8.63	2.91	0.83	9.15
Activation	PLI	63.82	1314.89	30.99		4.28	5.26
Activation	PLU	15.85	9.51	23.64		2.87	3.05
Activation	PTE	16.82	14.29	23.53		3.92	0.68
Activation	Liver activation fraction – dose 1	18.66	5.56	54.74		2.65	2.39	16.18
Activation	Liver activation fraction – dose 2	10.58	3.09	40.78		3.09	2.88	8.30
Activation	Lung activation fraction – dose 1	13.68	6.80	13.36		1.48	1.28
Activation	Lung activation fraction – dose 2	6.64	2.38	43.95	11.21	2.27	2.03
Activation	Testes activation fraction – dose 1	12.31	1.28	15.53		5.58	2.88
Activation	Testes activation fraction – dose 2	11.78	0.67	15.38		3.05	3.05

 

 

Table 5: Compound frequency summary report – suspension tests: with rat-derived metabolic preparations

 

 

Test	ORG	Test strain
		TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	3.73	2.82	5.33	1.49	2.48
Activation	Tissue control	6.20	20.67	9.50	3.82	3.65
Activation	Solvent control	4.15	3.33	4.84	1.61	1.93
Activation	PLI	38.29	819.28	19.72	4.20	4.06
Activation	PLU	5.79	7.73	13.56	5.54	3.23
Activation	PTE	5.65	3.07	10.07	4.09	1.57
Activation	Liver activation fraction – dose 1	7.86	1.49	8.47	3.68	1.70
Activation	Liver activation fraction – dose 2	6.53	2.16	7.78	2.50	1.48
Activation	Lung activation fraction – dose 1	5.67	5.45	10.48	3.09	2.26
Activation	Lung activation fraction – dose 2	8.93	5.28	9.21	2.39	2.39
Activation	Testes activation fraction – dose 1	6.77	10.05	10.25	3.16	0.61
Activation	Testes activation fraction – dose 2	7.89	11.00	13.04	2.15	0.89

 

 

Table 6: Compound frequency summary report – suspension tests: with monkey-derived metabolic preparations

 

 

Test	ORG	Test strain
		TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	18.01	9.85	11.55	1.35	1.35
Activation	Tissue control	15.28	7.78	16.84	2.32	1.93
Activation	Solvent control	10.14	9.39	10.94	0.70	0.17
Activation	PLI	672.50	31.09	62.27	7.67	6.39
Activation	PLU	20.28	18.71	6.06	3.63	1.68
Activation	PTE	9.66	8.87	19.29	1.36	2.87
Activation	Liver activation fraction – dose 1	14.45	11.54	9.09	1.24	1.99
Activation	Liver activation fraction – dose 2	9.60	14.91	13.54	1.57	1.46
Activation	Lung activation fraction – dose 1	14.45	11.54	9.09	1.24	1.99
Activation	Lung activation fraction – dose 2	18.51	8.41	6.30	3.46	2.80
Activation	Testes activation fraction – dose 1	15.79	9.05	15.93	2.60	1.36
Activation	Testes activation fraction – dose 2	10.19	7.96	16.72	2.84	1.84

Conclusions:

Monosodium phosphate anhydrous, powdered is considered to be non-mutagenic under the conditions of this study.

This study is considered to be sufficient to fulfil this endpoint as part of a weight of evidence and no further testing will be required for the following reasons:

As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting 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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA 1353, TA 1357, TA 1358

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: soluble under treatment conditions

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test date for toxicity determinations: March 25, 1975
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): 10.0, 1.0, 0.1, 0.01, 0.001.
-Concentrations of the test compound used in mutagenicity tests:

BACTERIA:
- 1/4 50% survival: 0.625%
-1/2 50% survival: 1.250%
-50% survival: 2.500%
-Plate tests: 1.250%

YEAST:
- 1/4 50% survival: 2.5%
-1/2 50% survival: 5.0%
-50% survival: 10.0%
-Plate tests: not applicable

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
Solvent control	-	-	40	41	11	13	27	33
Positive control^a	-	-	>10^3	>10^3	183	113	98	129
Test compound	-	-	44	46	8	14	15	22
2. Activation
Negative control	---	-	8	12	7	7	6	18
Solvent control		-	13	4	15	16	23	21
Reaction mixture control		-	7	10	8	8	10	18
Positive control^b	Mouse	Liver	>10^3	>10^3	41	43	307	420
Positive control		Lung	11	13	5	12	72	30
Positive control		Testes	9	11	22	10	19	22
Positive control	Rat	Liver	>10^3	>10^3	41	45	327	340
Positive control		Lung	12	9	7	7	26	29
Positive control		Testes	9	11	22	10	19	22
Positive control	Monkey	Liver	390	329	44	41	363	310
Positive control		Lung	11	9	7	10	21	24
Positive control		Testes	9	12	16	6	17	12
Test compound	Mouse	Liver	19	16	10	22	24	20
Test compound		Lung	23	20	7	5	17	21
Test compound		Testes	17	24	8	9	20	31
Test compound	Rat	Liver	16	14	10	14	23	15
Test compound		Lung	21	18	6	9	20	18
Test compound		Testes	17	22	7	10	21	29
Test compound	Monkey	Liver	19	15	15	24	20	14
Test compound		Lung	23	16	8	8	18	21
Test compound		Testes	21	20	7	8	19	29

 

^aTA 1353:. TA 1537: QM, TA 1538: NF

^bTA 1353: DMNA, TA 1537: AAF, TA 1538: AAF

 

 

Table 3: compound frequency summary report – suspension tests

 

Test	Test strain
	TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Nonactivation: solvent control	7.58	23.63	15.00	1.07	0.93
Nonactivation: positive control	376.09	688.06	666.27	2.08	117.36
Nonactivation: dose 1	13.02	14.14	10.00	1.81	1,27
Nonactivation: dose 2	11.71	14.62	8.08	2.74	1.46

 

 

Table 4: Compound frequency summary report – suspension tests: with mouse-derived metabolic preparations. The LI1, LI2 and LU2 dose levels were repeated with TA 1537 because of increased mutant frequencies. The repeat tests were negative.

 

Test	ORG	Test strain
		TA 1535	TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)	TA 1537
Activation	Negative chemical control	18.11	4.61	10.20		2.12	1.52
Activation	Tissue control	14.08	9.87	14.37		3.14	5.10
Activation	Solvent control	7.98	4.84	10.54	8.63	2.91	0.83	9.15
Activation	PLI	63.82	1314.89	30.99		4.28	5.26
Activation	PLU	15.85	9.51	23.64		2.87	3.05
Activation	PTE	16.82	14.29	23.53		3.92	0.68
Activation	Liver activation fraction – dose 1	18.66	5.56	54.74		2.65	2.39	16.18
Activation	Liver activation fraction – dose 2	10.58	3.09	40.78		3.09	2.88	8.30
Activation	Lung activation fraction – dose 1	13.68	6.80	13.36		1.48	1.28
Activation	Lung activation fraction – dose 2	6.64	2.38	43.95	11.21	2.27	2.03
Activation	Testes activation fraction – dose 1	12.31	1.28	15.53		5.58	2.88
Activation	Testes activation fraction – dose 2	11.78	0.67	15.38		3.05	3.05

 

 

Table 5: Compound frequency summary report – suspension tests: with rat-derived metabolic preparations

 

 

Test	ORG	Test strain
		TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	3.73	2.82	5.33	1.49	2.48
Activation	Tissue control	6.20	20.67	9.50	3.82	3.65
Activation	Solvent control	4.15	3.33	4.84	1.61	1.93
Activation	PLI	38.29	819.28	19.72	4.20	4.06
Activation	PLU	5.79	7.73	13.56	5.54	3.23
Activation	PTE	5.65	3.07	10.07	4.09	1.57
Activation	Liver activation fraction – dose 1	7.86	1.49	8.47	3.68	1.70
Activation	Liver activation fraction – dose 2	6.53	2.16	7.78	2.50	1.48
Activation	Lung activation fraction – dose 1	5.67	5.45	10.48	3.09	2.26
Activation	Lung activation fraction – dose 2	8.93	5.28	9.21	2.39	2.39
Activation	Testes activation fraction – dose 1	6.77	10.05	10.25	3.16	0.61
Activation	Testes activation fraction – dose 2	7.89	11.00	13.04	2.15	0.89

 

 

Table 6: Compound frequency summary report – suspension tests: with monkey-derived metabolic preparations

 

 

Test	ORG	Test strain
		TA 1535	TA 1537	TA 1538	D4 (ADE)	D4 (TRY)
Activation	Negative chemical control	18.01	9.85	11.55	1.35	1.35
Activation	Tissue control	15.28	7.78	16.84	2.32	1.93
Activation	Solvent control	10.14	9.39	10.94	0.70	0.17
Activation	PLI	672.50	31.09	62.27	7.67	6.39
Activation	PLU	20.28	18.71	6.06	3.63	1.68
Activation	PTE	9.66	8.87	19.29	1.36	2.87
Activation	Liver activation fraction – dose 1	14.45	11.54	9.09	1.24	1.99
Activation	Liver activation fraction – dose 2	9.60	14.91	13.54	1.57	1.46
Activation	Lung activation fraction – dose 1	14.45	11.54	9.09	1.24	1.99
Activation	Lung activation fraction – dose 2	18.51	8.41	6.30	3.46	2.80
Activation	Testes activation fraction – dose 1	15.79	9.05	15.93	2.60	1.36
Activation	Testes activation fraction – dose 2	10.19	7.96	16.72	2.84	1.84

Conclusions:

Trisodium phosphate is estimated to be non-mutagenic under the conditions of this study.

Executive summary:

Trisodium phosphate was estimated to be non-mutagenic as found in the source study performed with sodium phosphate, monobasic. As explained in the justification for type of information, the differences in molecular structure between trisodium phosphate and sodium phosphate, monobasic are unlikely to lead to differences in the genetic toxicity that are higher than the typical experimental error of the test method

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to 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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

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:

Only 4 strains of bacteria were studied. The guideline states that if the postive control 2-AA is used an additional control to determine metabolic activation by microsomal enzymes is required, the purity of the test substance is not determined

GLP compliance:

no

Remarks:

Study predates GLP

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Liver S-9 from Sprague Dawley rats (SRI) and Syrian hamsters (HLI)

Test concentrations with justification for top dose:

10, 33, 100, 3333, 10000 µg/plate

Vehicle / solvent:

- Vehicle)/solvent used: water

Untreated negative controls:

yes

Remarks:

sodium phosphate, dibasic is used in the assay as a negative control. Other negative controls: choline chloride, glycerol, glycine and mannitol

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

Sodium phosphate ,dibasic is known to be non-mutagenic, results also support this assumption. Other negative controls: choline chloride, glycerol, glycine and mannitol

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2-AA)

Remarks:

2-AA was tested on all strains in the presence of rat and hamster S-9

Untreated negative controls:

yes

Remarks:

sodium phosphate, dibasic is used in the assay as a negative control. Other negative controls: choline chloride, glycerol, glycine and mannitol

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

Sodium phosphate ,dibasic is known to be non-mutagenic, results also support this assumption. Other negative controls: choline chloride, glycerol, glycine and mannitol

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Remarks:

NOPD was tested on strain TA 98 without S-9

Untreated negative controls:

yes

Remarks:

sodium phosphate, dibasic is used in the assay as a negative control. Other negative controls: choline chloride, glycerol, glycine and mannitol

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

Sodium phosphate ,dibasic is known to be non-mutagenic, results also support this assumption. Other negative controls: choline chloride, glycerol, glycine and mannitol

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

sodium azide was tested on strains TA 100 and TA 1535 without S-9

Untreated negative controls:

yes

Remarks:

sodium phosphate, dibasic is used in the assay as a negative control. Other negative controls: choline chloride, glycerol, glycine and mannitol

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

Sodium phosphate ,dibasic is known to be non-mutagenic, results also support this assumption. Other negative controls: choline chloride, glycerol, glycine and mannitol

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

9-amino acridine was tested on strain TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C
- Expression time (cells in growth medium): 12 -15 hours at 37°C

DETERMINATION OF CYTOTOXICITY
- Method: One or more parameters were used as an indication of toxicity: viability on complete medium (EGG studies) and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn (CWR, EGG, SRI studies)

Evaluation criteria:

Statistical analysis was not incorporated into the initial data evaluations. The data were evaluated in an ad hoc manner by each testing laboratory and by NTP personnel. Prior to statistical analysis no formal rules were used; however, a positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not. The matrix of test strains and activation systems used allowed the investigators to detect trends or patterns that might not be as evident if only one strain and activation system, were examined. In addition to the standard ‘positive’ and ‘negative’ categories, there is also ‘questionable’ or ‘inconclusive’. This applied to low-level responses that were not reproducible within the laboratory or to results that showed a definite trend but with which the investigator did not feel comfortable in making a ‘+’ or ‘-‘ decision. It also included tests in which an elevated revertant colony yield occurred at only a single dose level.

Statistics:

Data were subsequently evaluated using analysis based on the models presented by Margolin et al (1981); this analysis is described elsewhere (Risko et al, in preparation).

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

Vehicle controls validity:

valid

Untreated negative controls validity:

other: all negative controls were valid with the exception of streptomycin sulphate which was mutagenic in tester strain TA 98 in 2 out of 3 of the testing laboratories and glycerol with gave equivocal results

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: distilled water was chosen as a solvent as the substance is soluble in water

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Tables 2 -6: The results presented below are from the most definitive experiment conducted on each chemical, in this case sodium phosphate, dibasic, (usually the results from the confirmation assay are considered to be definitive). Results are presented as mean ± SEM

 

Table 2: Study conducted at CWR, vehicle unknown.

 

Dose (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

92 ± 4.5

128 ± 2.7

151 ± 8.0

8 ± 1.5

15 ± 2.0

9 ± 2.0

8 ± 1.2

6 ± 0.6

8 ± 0.7

13 ± 0.3

14 ± 2.7

18 ± 1.2

10.0

88 ± 3.2

112 ± 7.0

182 ± 49.5

8 ± 1.5

16 ± 1.7

8 ± 1.5

7 ± 1.5

7 ± 0.7

6 ± 0.3

16 ± 3.0

12 ± 0.3

16 ± 0.9

33.0

90 ± 3.1

110 ± 7.4

146 ± 2.0

9 ± 0.9

11 ± 2.2

11 ± 1.5

6 ± 1.0

7 ± 1.8

5 ± 0.5

17 ± 1.8

11 ± 0.3

16 ± 2.0

100.0

109 ± 22.4

124 ± 10.1

126 ± 4.7

11 ± 1.7

8 ± 1.7

8 ± 2.0

5 ± 0.3

8 ± 0.6

7 ± 1.5

14 ± 4.8

19 ± 0.9

13 ± 3.8

3333.0

84 ± 5.2

117 ± 6.4

139 ± 4.4

4 ± 0.9

12 ± 1.7

10 ± 2.0

8 v 0.5

8 ± 2.3

6 ± 0.3

12 ± 0.7

19 ± 2.0

15 ± 3.4

10000.0

77 ± 4.6

139 ± 11.3

147 ± 7.8

8 ± 0.0

12 ± 2.0

13 ± 1.3

6 ± 1.5

8 ± 0.3

8 ± 1.8

14 ± 1.3

13 ± 1.5

14 ± 2.8

POS^1

367 ± 35.8

904 ± 165.7

1275 ± 147.0

255 ± 18.0

44 ± 9.7

69 ± 6.2

214 ± 26.9

31 ± 2.3

60 ± 11.7

264 ± 26.5

151 ± 17.0

479 ± 69.5

 

Table 3: Study conducted at CWR, vehicle: water

 

Dose   (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

121 ± 2.2

133 ± 5.9

136 ± 5.2

5 ± 0.3

7 ± 1.3

7 ± 1.5

3 ± 0.7

11 ± 1.5

8 ± 0.9

13 ± 2.5

20 ± 0.3

18 ± 3.8

10.0

115 ± 6.5

138 ± 8.1

120 ± 6.7

4 ± 0.9

7 ± 0.7

8 ± 1.0

2 ± 0.7

9 ± 0.3

6 ± 2.0

9 ± 1.9

20 ± 2.7

20 ± 3.7

33.0

93 ± 4.6

141 ± 11.4

131 ± 5.1

6 ± 2.6

5 ± 0.9

6 ± 0

4 ± 1.2

9 ± 1.5

8 ± 1.9

8 ± 2.1

13 ± 2.0

14 ± 2.6

100.0

103 ± 4.0

113 ± 5.8

122 ± 7.9

6 ± 1.5

6 ± 2.3

2 ± 0.3

2 ± 0.6

6 ± 1.2

6 ± 0.7

8 ± 2.5

16 ± 2.3

16 ± 0.6

3333.0

95 ± 4.5

124 ± 4.3

131 ± 2.9

7 ± 1.2

7 ± 1.5

7 ± 2.2

4 ± 1.7

8 ± 1.0

9 ± 0.9

11 ± 0.9

20 ± 3.8

16 ± 0.6

10000.0

103 ± 5.2

123 ± 3.5

131 ± 6.4

7 ± 2.7

7 ± 1.5

7 ± 1.7

3 ± 0.3

6 ± 1.5

8 ± 2.0

11 ± 1.2

18 ± 4.0

17 ± 0.7

POS^1

585 ± 40.5

1001 ± 112.9

1915 ± 139.3

623 ± 19.8

51 ± 0.7

160 ± 11.1

247 ± 99.6

73 ± 11.8

38 ± 3.0

234 ± 36.7

836 ± 161.8

1442 ± 86.7

 

Table 4: Study conducted at EGG, vehicle: water

 

Dose  (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

149 ± 2.7

137 ± 10.3

116 ± 4.8

37 ± 3.6

11 ± 1.8

14 ± 2.5

8 ± 1.5

12 ± 2.2

11 ± 1.5

15 ± 1.2

27 v±1.9

31 ± 2.1

10.0

137 ± 12.0

125 ± 2.8

115 ± 3.5

31 ± 3.2

16 ± 1.9

14 ± 1.3

8 ± 1.5

10 ± 0.0

8 ± 1.2

21 ± 0.7

27 ± 3.4

26 ± 2.3

33.0

138 ± 5.6

128 ± 5.5

115 ± 9.7

30 ± 1.7

12 ± 2.0

9 ± 0.6

10 ± 1.5

7 ± 1.5

8 ± 0.9

20 ± 2.2

32 ± 2.5

30 ± 2.9

100.0

144 ± 10.5

120 ± 4.3

125 ± 9.2

31 ± 3.2

14 ± 2.9

11 ± 2.6

5 ± 0.6

11 ± 0.7

8 ± 1.5

16 ± 1.2

32 ± 1.8

25 ± 2.4

3333.0

137 ± 3.0

116 ± 11.5

115 ± 8.0

32 ± 4.5

15 ± 2.5

11 ± 0.6

9 ± 2.5

11 ± 2.1

14 ± 3.2

25 ± 2.5

32 ± 3.3

30 ± 0.7

10000.0

138 ± 4.4

116 ± 11.3

103 ± 9.0

30 ± 3.1

14 ± 2.6

13 ± 1.3

9 ± 0.0

10 ± 1.5

10 ± 2.0

21 ± 1.3

25 ± 1.9

31 ± 2.3

POS^1

861 ± 28.7

852 ± 31.5

1126 ± 50.8

737 ± 13.2

75 ± 5.9

101 ± 2.6

282 ± 95.0

76 ± 3.6

104 ± 5.9

1161 ± 20.0

710 ± 48.1

995 ± 32.9

 

  Table 5: Study conducted at SRI, vehicle: water

 

Dose  (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

79 ± 0.6

91 ± 7.0

134 ± 4.0

13 ± 0.9

6 ± 0.3

18 ± 1.0

13 ± 3.0

13 ± 2.6

9 ± 1.5

15 ± 1.7

18 ± 1.8

18 ± 3.2

10.0

92 ± 6.0

103 ± 2.4

90 ± 7.8

16 ± 1.0

10 ± 2.6

6 ± 0.3

10 ± 0.9

12 ± 2.0

8 ± 0.7

15 ± 3.1

26 ± 5.5

20 ± 0.9

33.0

80 ± 3.5

96 ± 9.3

92 ± 3.0

11 ± 1.8

8 ± 3.5

5 ± 0.3

10 ± 1.8

17 ± 2.0

10 ± 0.9

17 ± 2.0

26 ± 3.5

17 ± 2.2

100.0

84 ± 3.7

8 7± 7.7

83 ± 8.5

12 ± 2.5

6 ± 0.3

5 ± 1.2

8 ± 1.2

16 ± 2.4

10 ± 1.2

14 ± 0.7

20 ± 0.6

22 ± 1.7

3333.0

82 ± 4.1

96 ± 1.7

91 ± 6.1

10 ± 1.9

9 ± 3.1

4 ± 1.0

7 ± 0.7

13 ± 3.2

10 ± 3.2

12 ± 0.9

21 ± 5.5

23 ± 7.3

10000.0

88 ± 5.4

86 ± 4.0

103 ± 6.4

13 ± 0.3

7 ± 0.1

9 ± 0.0

9 ± 1.7

10 ± 3.0

10 ± 1.5

15 ± 0.9

11 ± 1.2

20 ± 3.4

POS^1

458 ± 10.7

553 ± 5.5

11621 ± 10.3

375 ±4.9

271 ± 15.5

315 ± 3.8

63 ± 7.8

311 ± 10.5

348 ± 4.0

498 ± 19.7

320 ± 18.8

864 ± 46.7

 

 

Table 6: Study conducted at SRI, vehicle: water

 

Dose  (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

159 ± 7.4

158 ± 4.1

155 ± 2.9

32 ± 3.0

16 ± 1.5

15 ± 3.2

13 ± 0.7

16 ± 1.9

18 ± 1.0

30 ± 1.5

44 ± 3.7

42 ± 1.7

10.0

174 ± 4.8

154 ± 22.1

160 ± 3.2

26 ± 2.4

14 ± 2.6

16 ± 1.5

10 ± 2.2

23 ± 2.8

17 ± 1.2

31 ± 0.9

47 ± 2.0

51 ± 4.4

33.0

181 ± 3.8

149 ± 6.7

164 ± 10.5

24 ± 1.7

12 ± 1.9

19 ± 1.5

7 ± 1.2

18 ± 1.3

13 ± 2.2

31 ± 2.6

50 ± 10.0

62 ± 5.2

100.0

178 ± 12.2

158 ± 8.0

161 ± 11.0

26 ± 2.7

17 ± 1.5

18 ± 2.5

11 ± 4.2

15 ± 1.5

17 ± 1.2

28 ± 1.5

42 ± 7.2

74 ± 1.5

3333.0

181 ± 10.3

135 ± 3.9

160 ± 7.2

22 ± 5.0

17 ± 1.8

16 ± 1.5

12 ± 0.3

17 ± 2.2

17 ± 2.1

37 ± 2.0

40 ± 5.9

48 ± 3.2

10000.0

180 ± 15.7

155 ± 5.0

162 ± 11.3

24 ± 4.0

17 ± 1.2

20 ± 3.5

13 ± 4.1

16 ± 2.0

16 ± 5.6

29 ± 1.7

39 ± 2.4

51 ± 0.9

POS^1

471 ± 17.5

1262 ± 54.6

158 ± 55.1

359 ± 12.5

438 ± 30.3

505 ± 13.2

215 ± 8.4

491 ± 60.8

559 ± 5.5

815 ± 4.9

1013 ± 178.1

1393 ± 120.7

 

^1.Positive control

 

Conclusions:

Sodium phosphate, dibasic was used as a negative control in the reported assay. The results of the assay confirm that the substance is not genotoxic to S. typhimurium under the conditions reported.

This study is considered to be sufficient to fulfil this endpoint as part of a weight of evidence and no further testing will be required for the following reasons:

As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting 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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: all negative controls were valid with the exception of streptomycin sulphate which was mutagenic in tester strain TA 98 in 2 out of 3 of the testing laboratories and glycerol with gave equivocal results

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: distilled water was chosen as a solvent as the substance is soluble in water

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Tables 2 -6: The results presented below are from the most definitive experiment conducted on each chemical, in this case sodium phosphate, dibasic, (usually the results from the confirmation assay are considered to be definitive). Results are presented as mean ± SEM

 

Table 2: Study conducted at CWR, vehicle unknown.

 

Dose (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

92 ± 4.5

128 ± 2.7

151 ± 8.0

8 ± 1.5

15 ± 2.0

9 ± 2.0

8 ± 1.2

6 ± 0.6

8 ± 0.7

13 ± 0.3

14 ± 2.7

18 ± 1.2

10.0

88 ± 3.2

112 ± 7.0

182 ± 49.5

8 ± 1.5

16 ± 1.7

8 ± 1.5

7 ± 1.5

7 ± 0.7

6 ± 0.3

16 ± 3.0

12 ± 0.3

16 ± 0.9

33.0

90 ± 3.1

110 ± 7.4

146 ± 2.0

9 ± 0.9

11 ± 2.2

11 ± 1.5

6 ± 1.0

7 ± 1.8

5 ± 0.5

17 ± 1.8

11 ± 0.3

16 ± 2.0

100.0

109 ± 22.4

124 ± 10.1

126 ± 4.7

11 ± 1.7

8 ± 1.7

8 ± 2.0

5 ± 0.3

8 ± 0.6

7 ± 1.5

14 ± 4.8

19 ± 0.9

13 ± 3.8

3333.0

84 ± 5.2

117 ± 6.4

139 ± 4.4

4 ± 0.9

12 ± 1.7

10 ± 2.0

8 v 0.5

8 ± 2.3

6 ± 0.3

12 ± 0.7

19 ± 2.0

15 ± 3.4

10000.0

77 ± 4.6

139 ± 11.3

147 ± 7.8

8 ± 0.0

12 ± 2.0

13 ± 1.3

6 ± 1.5

8 ± 0.3

8 ± 1.8

14 ± 1.3

13 ± 1.5

14 ± 2.8

POS^1

367 ± 35.8

904 ± 165.7

1275 ± 147.0

255 ± 18.0

44 ± 9.7

69 ± 6.2

214 ± 26.9

31 ± 2.3

60 ± 11.7

264 ± 26.5

151 ± 17.0

479 ± 69.5

 

Table 3: Study conducted at CWR, vehicle: water

 

Dose   (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

121 ± 2.2

133 ± 5.9

136 ± 5.2

5 ± 0.3

7 ± 1.3

7 ± 1.5

3 ± 0.7

11 ± 1.5

8 ± 0.9

13 ± 2.5

20 ± 0.3

18 ± 3.8

10.0

115 ± 6.5

138 ± 8.1

120 ± 6.7

4 ± 0.9

7 ± 0.7

8 ± 1.0

2 ± 0.7

9 ± 0.3

6 ± 2.0

9 ± 1.9

20 ± 2.7

20 ± 3.7

33.0

93 ± 4.6

141 ± 11.4

131 ± 5.1

6 ± 2.6

5 ± 0.9

6 ± 0

4 ± 1.2

9 ± 1.5

8 ± 1.9

8 ± 2.1

13 ± 2.0

14 ± 2.6

100.0

103 ± 4.0

113 ± 5.8

122 ± 7.9

6 ± 1.5

6 ± 2.3

2 ± 0.3

2 ± 0.6

6 ± 1.2

6 ± 0.7

8 ± 2.5

16 ± 2.3

16 ± 0.6

3333.0

95 ± 4.5

124 ± 4.3

131 ± 2.9

7 ± 1.2

7 ± 1.5

7 ± 2.2

4 ± 1.7

8 ± 1.0

9 ± 0.9

11 ± 0.9

20 ± 3.8

16 ± 0.6

10000.0

103 ± 5.2

123 ± 3.5

131 ± 6.4

7 ± 2.7

7 ± 1.5

7 ± 1.7

3 ± 0.3

6 ± 1.5

8 ± 2.0

11 ± 1.2

18 ± 4.0

17 ± 0.7

POS^1

585 ± 40.5

1001 ± 112.9

1915 ± 139.3

623 ± 19.8

51 ± 0.7

160 ± 11.1

247 ± 99.6

73 ± 11.8

38 ± 3.0

234 ± 36.7

836 ± 161.8

1442 ± 86.7

 

Table 4: Study conducted at EGG, vehicle: water

 

Dose  (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

149 ± 2.7

137 ± 10.3

116 ± 4.8

37 ± 3.6

11 ± 1.8

14 ± 2.5

8 ± 1.5

12 ± 2.2

11 ± 1.5

15 ± 1.2

27 v±1.9

31 ± 2.1

10.0

137 ± 12.0

125 ± 2.8

115 ± 3.5

31 ± 3.2

16 ± 1.9

14 ± 1.3

8 ± 1.5

10 ± 0.0

8 ± 1.2

21 ± 0.7

27 ± 3.4

26 ± 2.3

33.0

138 ± 5.6

128 ± 5.5

115 ± 9.7

30 ± 1.7

12 ± 2.0

9 ± 0.6

10 ± 1.5

7 ± 1.5

8 ± 0.9

20 ± 2.2

32 ± 2.5

30 ± 2.9

100.0

144 ± 10.5

120 ± 4.3

125 ± 9.2

31 ± 3.2

14 ± 2.9

11 ± 2.6

5 ± 0.6

11 ± 0.7

8 ± 1.5

16 ± 1.2

32 ± 1.8

25 ± 2.4

3333.0

137 ± 3.0

116 ± 11.5

115 ± 8.0

32 ± 4.5

15 ± 2.5

11 ± 0.6

9 ± 2.5

11 ± 2.1

14 ± 3.2

25 ± 2.5

32 ± 3.3

30 ± 0.7

10000.0

138 ± 4.4

116 ± 11.3

103 ± 9.0

30 ± 3.1

14 ± 2.6

13 ± 1.3

9 ± 0.0

10 ± 1.5

10 ± 2.0

21 ± 1.3

25 ± 1.9

31 ± 2.3

POS^1

861 ± 28.7

852 ± 31.5

1126 ± 50.8

737 ± 13.2

75 ± 5.9

101 ± 2.6

282 ± 95.0

76 ± 3.6

104 ± 5.9

1161 ± 20.0

710 ± 48.1

995 ± 32.9

 

  Table 5: Study conducted at SRI, vehicle: water

 

Dose  (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

79 ± 0.6

91 ± 7.0

134 ± 4.0

13 ± 0.9

6 ± 0.3

18 ± 1.0

13 ± 3.0

13 ± 2.6

9 ± 1.5

15 ± 1.7

18 ± 1.8

18 ± 3.2

10.0

92 ± 6.0

103 ± 2.4

90 ± 7.8

16 ± 1.0

10 ± 2.6

6 ± 0.3

10 ± 0.9

12 ± 2.0

8 ± 0.7

15 ± 3.1

26 ± 5.5

20 ± 0.9

33.0

80 ± 3.5

96 ± 9.3

92 ± 3.0

11 ± 1.8

8 ± 3.5

5 ± 0.3

10 ± 1.8

17 ± 2.0

10 ± 0.9

17 ± 2.0

26 ± 3.5

17 ± 2.2

100.0

84 ± 3.7

8 7± 7.7

83 ± 8.5

12 ± 2.5

6 ± 0.3

5 ± 1.2

8 ± 1.2

16 ± 2.4

10 ± 1.2

14 ± 0.7

20 ± 0.6

22 ± 1.7

3333.0

82 ± 4.1

96 ± 1.7

91 ± 6.1

10 ± 1.9

9 ± 3.1

4 ± 1.0

7 ± 0.7

13 ± 3.2

10 ± 3.2

12 ± 0.9

21 ± 5.5

23 ± 7.3

10000.0

88 ± 5.4

86 ± 4.0

103 ± 6.4

13 ± 0.3

7 ± 0.1

9 ± 0.0

9 ± 1.7

10 ± 3.0

10 ± 1.5

15 ± 0.9

11 ± 1.2

20 ± 3.4

POS^1

458 ± 10.7

553 ± 5.5

11621 ± 10.3

375 ±4.9

271 ± 15.5

315 ± 3.8

63 ± 7.8

311 ± 10.5

348 ± 4.0

498 ± 19.7

320 ± 18.8

864 ± 46.7

 

 

Table 6: Study conducted at SRI, vehicle: water

 

Dose  (µg/plate)

Strain: TA 100

Strain: TA 1535

Strain: TA 1537

Strain: TA 98

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

No S-9

RLI

HLI

0.0

159 ± 7.4

158 ± 4.1

155 ± 2.9

32 ± 3.0

16 ± 1.5

15 ± 3.2

13 ± 0.7

16 ± 1.9

18 ± 1.0

30 ± 1.5

44 ± 3.7

42 ± 1.7

10.0

174 ± 4.8

154 ± 22.1

160 ± 3.2

26 ± 2.4

14 ± 2.6

16 ± 1.5

10 ± 2.2

23 ± 2.8

17 ± 1.2

31 ± 0.9

47 ± 2.0

51 ± 4.4

33.0

181 ± 3.8

149 ± 6.7

164 ± 10.5

24 ± 1.7

12 ± 1.9

19 ± 1.5

7 ± 1.2

18 ± 1.3

13 ± 2.2

31 ± 2.6

50 ± 10.0

62 ± 5.2

100.0

178 ± 12.2

158 ± 8.0

161 ± 11.0

26 ± 2.7

17 ± 1.5

18 ± 2.5

11 ± 4.2

15 ± 1.5

17 ± 1.2

28 ± 1.5

42 ± 7.2

74 ± 1.5

3333.0

181 ± 10.3

135 ± 3.9

160 ± 7.2

22 ± 5.0

17 ± 1.8

16 ± 1.5

12 ± 0.3

17 ± 2.2

17 ± 2.1

37 ± 2.0

40 ± 5.9

48 ± 3.2

10000.0

180 ± 15.7

155 ± 5.0

162 ± 11.3

24 ± 4.0

17 ± 1.2

20 ± 3.5

13 ± 4.1

16 ± 2.0

16 ± 5.6

29 ± 1.7

39 ± 2.4

51 ± 0.9

POS^1

471 ± 17.5

1262 ± 54.6

158 ± 55.1

359 ± 12.5

438 ± 30.3

505 ± 13.2

215 ± 8.4

491 ± 60.8

559 ± 5.5

815 ± 4.9

1013 ± 178.1

1393 ± 120.7

 

^1.Positive control

 

Conclusions:

Trisodium phosphate is estimated to be non-mutagenic under the conditions of this study.

Executive summary:

Trisodium phosphate was estimated to be non-mutagenic as found in the source study performed with disodium phosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium phosphate and disodium phosphate, monobasic are unlikely to lead to differences in the genetic toxicity that are higher than the typical experimental error of the test method

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

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 positive control data

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Remarks:

Study predates GLP

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium, other: TA 92, TA 1535, TA 100, TA1537, 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: 100.0 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffer

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Test strains exposed to the appropriate solvent or untreated

True negative controls:

not specified

Positive controls:

not specified

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, TA1537, TA 94 and TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

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 literature:

Additive	Max dose (mg/plate)	Result
Sodium phosphate, monobasic	100.0	Negative

Conclusions:

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

This study is considered to be sufficient to fulfil this endpoint as part of a weight of evidence and no further testing will be required for the following reasons:

As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting 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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA 92, TA 1535, TA 100, TA1537, TA 94 and TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

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 literature:

Additive	Max dose (mg/plate)	Result
Sodium phosphate, monobasic	100.0	Negative

Conclusions:

Trisodium phosphate is estimated to be non-mutagenic under the conditions of this study.

Executive summary:

Trisodium phosphate was estimated to be non-mutagenic as found in the source study performed with monosodium phosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium phosphate and monosodium phosphate are unlikely to lead to differences in the genetic toxicity that are higher than the typical experimental error of the test method

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Study does not cover all tester strains recommended in the guideline. Positive control substances differ from those recommended in the guideline

Principles of method if other than guideline:

In addition to the bacterial studies, the same assay was also performed on Saccharomyces cerevisiae, strain: D4.

GLP compliance:

not specified

Remarks:

Study predates GLP

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538 TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

yeast, other: Saccharomyces cerevisiae, D4

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 homogenate prepared from Sprague-Dawley adult male rat liver induced by Aroclor 1254 5-days prior to kill.

Test concentrations with justification for top dose:

0.001, 0.01, 0.1, 1.0 and 5 μL per plate

Vehicle / solvent:

- Vehicle/solvent used: distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: methylnitrosoguanidine (10 µg/plate)

Remarks:

Used with tester strains TA 1535, TA 100 and S. cerevisiae; D4, in assays without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: quinacrine mustard (10 µg/plate)

Remarks:

Used with tester strains TA 1537, in assays without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Used with tester strains TA 1538 and TA 98, in assays without metabolic activation Migrated to IUCLID6: 100 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-anthramine (100 µg/plate)

Remarks:

Used with tester strains TA 1535 and TA 100, in assays with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 8-aminoquinoline (100 µg/plate)

Remarks:

Used with tester strains TA 1537, in assays with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

Used with tester strains TA 1538 and TA 98, in assays with metabolic activation Migrated to IUCLID6: 100 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: DMNA (100 µMol/plate)

Remarks:

Used with tester strains S. cerevisiae; D4, in assays with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

DURATION
- Exposure duration: 48 hr at 37°C
- Expression time (cells in growth medium): overnight

Evaluation criteria:

The criteria used to determine positive effects are inherently subjective and are based on historical data. Most data sets were evaluated using the following criteria:

- STRAINS TA 1353, TA-1357 and TA 1358
If the solvent control value is within the normal range, a chemical that produces a positive dose response over 3 concentrations with the lowest increase equal to 2x the solvent control value is considered to be mutagenic (positive result).

- STRAINS TA 98, TA 100 AND D4:
If the solvent control value is within the normal range, a chemical that produces a positive response of 3 concentrations with the highest increase equal to 2x the solvent value (TA 100) and 2-3x the solvent control value (TA 98 and D4) is considered to be mutagenic (positive result). The dose-response increase should start at approximately the solvent control value.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

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:

valid

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-258

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	10		11		15		32		174		70
	0.01	10		12		10		29		237		56
	0.1	12		11		7		27		187		54
	1.0	13		8		9		25		233		56
	5.0	13		5		7		22		179		59
Activation
Solvent control	-	13		16		16		43		124		35
Positive control	***	242		208		428		650		>1000		82
Test compound	0.001	18		23		16		35		146		45
	0.01	23		21		19		33		161		55
	0.1	28		16		11		41		182		56
	1.0	27		20		21		45		145		53
	5.0	29		30		7		41		193		58

* 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-256 did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered not-mutagenic under these test conditions.

This study is considered to be sufficient to fulfil this endpoint as part of a weight of evidence and no further testing will be required for the following reasons:

As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting 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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

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:

valid

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-258

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	10		11		15		32		174		70
	0.01	10		12		10		29		237		56
	0.1	12		11		7		27		187		54
	1.0	13		8		9		25		233		56
	5.0	13		5		7		22		179		59
Activation
Solvent control	-	13		16		16		43		124		35
Positive control	***	242		208		428		650		>1000		82
Test compound	0.001	18		23		16		35		146		45
	0.01	23		21		19		33		161		55
	0.1	28		16		11		41		182		56
	1.0	27		20		21		45		145		53
	5.0	29		30		7		41		193		58

* 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:

Trisodium phosphate is estimated to be non-mutagenic under the conditions of this study.

Executive summary:

Trisodium phosphate was estimated to be non-mutagenic as found in the source study performed with MCTR-256 As explained in the justification for type of information, the differences in molecular structure between trisodium phosphate and MCTR-256 are unlikely to lead to differences in the genetic toxicity that are higher than the typical experimental error of the test method

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Study does not cover all tester strains recommended in the guideline. Positive control substances differ from those recommended in the guideline

Principles of method if other than guideline:

In addition to the bacterial studies, the same assay was also performed on Saccharomyces cerevisiae, strain: D4.

GLP compliance:

not specified

Remarks:

Study predates GLP

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538 TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

yeast, other: Saccharomyces cerevisiae, D4

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 homogenate prepared from Sprague-Dawley adult male rat liver induced by Aroclor 1254 5-days prior to kill.

Test concentrations with justification for top dose:

0.001, 0.01, 0.1, 1.0 and 5 μL per plate

Vehicle / solvent:

- Vehicle/solvent used: deionized 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:

valid

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:

valid

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 an AMES study on MCTR-258

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		15		30		154		59
Positive control	**	236		901		408		721		530		>1000
Test compound	0.001	15		9		10		25		134		63
	0.01	8		6		7		29		167		40
	0.1	8		12		14		22		257		47
	1.0	10		9		10		20		172		40
	5.0	14		9		7		26		224		75
Activation
Solvent control	-	13		22		16		43		124		35
Positive control	***	242		208		428		650		>1000		82
Test compound	0.001	13		23		17		35		175		45
	0.01	19		31		8		41		148		48
	0.1	20		12		12		40		136		38
	1.0	17		22		12		38		124		45
	5.0	24		29		10		20		156		45

* 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-258 did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered not-mutagenic under these test conditions.

This study is considered to be sufficient to fulfil this endpoint as part of a weight of evidence and no further testing will be required for the following reasons:

As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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.

Reference 12

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting 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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

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:

valid

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 an AMES study on MCTR-258

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		15		30		154		59
Positive control	**	236		901		408		721		530		>1000
Test compound	0.001	15		9		10		25		134		63
	0.01	8		6		7		29		167		40
	0.1	8		12		14		22		257		47
	1.0	10		9		10		20		172		40
	5.0	14		9		7		26		224		75
Activation
Solvent control	-	13		22		16		43		124		35
Positive control	***	242		208		428		650		>1000		82
Test compound	0.001	13		23		17		35		175		45
	0.01	19		31		8		41		148		48
	0.1	20		12		12		40		136		38
	1.0	17		22		12		38		124		45
	5.0	24		29		10		20		156		45

* 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:

Trisodium phosphate is estimated to be non-mutagenic under the conditions of this study.

Executive summary:

Trisodium phosphate was estimated to be non-mutagenic as found in the source study performed with MCTR-258. As explained in the justification for type of information, the differences in molecular structure between trisodium phosphate and MCTR-258 are unlikely to lead to differences in the genetic toxicity that are higher than the typical experimental error of the test method

Reference 13

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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

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.

Reference 14

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In preliminary experiment, cytotoxicity was noted at a concentration of 1000 or 2000 ug/mL in the presence of metabolic activation (3-hour exposure)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Results
A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation.A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000, and 2000 µg sodium dihydrogenorthophosphate/mL medium were tested for cytotoxicity. Cytotoxicity (decreased survival) was noted at a concentration of 1000 or 2000 µg/mL in the presence of metabolic activation (3-hour exposure). For details, seeTable 1. No changes in pH or osmolality were noted in the test item formulations compared to the control (seeText table 7-1 'pH values and osmolality').

Hence, in the main study the highest concentration employed was 2000 µg sodium dihydrogenorthophosphate/mL medium in the experiments without and with metabolic activation.

Methylmethanesulfonate (0.013 or 0.012 µg/mL for a 3- and 24-hour exposure, respectively) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (1.0 µg/mL) in the presence of exogenous metabolic activation.

In the main study, cytotoxicity (decreased relative total growth) was noted in the first experiment with metabolic activation at concentrations of 1000 and 2000 µg sodium dihydrogenorthophosphate/mL medium and at 2000 µg/mL medium in the second experiment with S9. No signs of cytotoxicity were noted in the experiments without metabolic activation.

The negative controls had mutation frequencies of 63.89 or 137.93 per 106 clonable cells in the experiments without metabolic activation (3- or 24-hour exposure, respectively, for details seeTable 3a) and 55.47 or 120.71 per 106 clonable cells in the experiments with metabolic activation (for details see Table 3b) and, hence, were all well within the historical data-range.

The mutation frequencies of the cultures treated with sodium dihydrogenorthophosphate ranged from 57.17 to 74.73 per 106 clonable cells (3-hour exposure) and from 84.33 to 164.59 per 106clonable cells (24-hour exposure) in the experiments without metabolic activation (for details seeTable 3a). In the experiments with metabolic activation, mutation frequencies ranged from 63.82 to 86.21 per 106 clonable cells (3-hour exposure, first assay) and from 50.23 to 155.35 per 106 clonable cells (3-hour exposure, second assay) (for details, seeTable 3b). These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.40to 1.31 for sodium dihydrogenorthophosphate-treated cells and ratios of 0.42 to 0.94for the negative controls (see Table 4a and Table 4b).

The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3‑MC) caused pronounced increases in the mutation frequency of 474.25 and 652.55 per 106clonable cells in the case of MMS (for details seeTable 3a) and of 387.42 and 489.98 per 106clonable cells in the case of 3-MC (for details seeTable 3b). All positive controls showed an increase in the small colony MF of at least 150 x 10-6above that seen in the concurrent solvent control and an absolute increase in total mutation frequency of at least 300 x 10-6. Furthermore, the mean relative total growth (RTG) for the positive controls was greater than or equal to 10%. Hence, the acceptance criteria were met.

The calculations of suspension growth of the negative controls were in the acceptance criteria range between 8 and 32 following 3-hour treatments or between 32 and 180 following 24-hour treatments (for details see Table 2a and Table 2b). The mean cloning efficiencies (CE = PEx 100) of the negative controls from the Mutation Experiments were between the range 65% to 120% (see Table 3a and Table 3b). Hence, the acceptance criteria described in were met.

The mutation frequency and the colony size ratio of the positive controls and negative controls without and with metabolic activation for the last 11 experiments (background data, not audited by the QAU-department) are attached.

The pH and osmolality data of the negative control and of all test item formulations in the medium were determined in the first preliminary test - see Text table 7-1 below in 'Any other information on results incl. tables.'
 No relevant changes in pH or osmolality of the test item formulations at concentrations of 10.0 to 2000 µg/mL medium compared to the negative control were noted.

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:

Trisodium phosphate is estimated to be non-genotoxic in a L5178Y TK +/- mammalian cell mutagenicity test as found in the source study performed with sodium dihydrogenorthophosphate.

Executive summary:

Trisodium phosphate is estimated to be non-genotoxic in a L5178Y TK +/- mammalian cell mutagenicity test as found in the source study performed with sodium dihydrogenorthophosphate. As explained in the justification for type of information, the differences in molecular structure between trisodium phosphate and sodium dihydrogenorthophosphate

are unlikely to lead to differences in the genotoxicity that are higher than the typical experimental error of the test method.

Reference 15

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 26 May 2010 and 24 June 2010.

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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully miscible in sterile distilled water at 50 mg/ml in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

Test Procedure
Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA-), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate).
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test material formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
The positive and untreated controls were dosed using the standard plate incorporation method described in Section "Mutation Test - Experiment 1".
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Evaluation criteria:

Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2008 and 2009 in Appendix 2.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges for 2008 and 2009 are presented in the attached Appendix 2.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS
Preliminary Toxicity Test
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay were:


With (+) or without (-)
S9-mix Strain Dose (µg/plate)
0 0.15 0.5 1.5 5 15 50 150 500 1500 5000
- TA100 88 80 111 93 98 86 90 93 76 99 74
+ TA100 98 74 102 88 109 92 80 114 97 90 99
- WP2uvrA- 22 27 18 23 24 19 22 25 21 22 22
+ WP2uvrA- 19 32 30 31 22 23 25 24 22 20 23

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.
A history profile of vehicle and positive control values for 2008 and 2009 is presented in Appendix 2.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of theS9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table1              Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
96		20		22		16		7
95	(93)	21	(20)	18	(21)	21	(21)	12	(11)
88		18		22		25		13

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
89		12		53		18		10
96	(97)	18	(13)	34	(40)	21	(21)	13	(11)
107		10		32		23		11

Table 2              Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 08 June 2010						To: 11 June 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA-			TA98		TA1537
-	0	108 92 89	(96) 10.2#	22 29 25	(25) 3.5	23 32 27	(27) 4.5		22 15 27	(21) 6.0	16 14 10	(13) 3.1
-	50	86 99 115	(100) 14.5	26 27 27	(27) 0.6	33 29 27	(30) 3.1		14 21 14	(16) 4.0	10 15 16	(14) 3.2
-	150	107 111 97	(105) 7.2	20 25 24	(23) 2.6	24 30 24	(26) 3.5		20 18 15	(18) 2.5	15 16 13	(15) 1.5
-	500	98 103 102	(101) 2.6	29 21 23	(24) 4.2	32 26 30	(29) 3.1		20 19 18	(19) 1.0	12 7 15	(11) 4.0
-	1500	103 103 117	(108) 8.1	23 20 18	(20) 2.5	27 31 26	(28) 2.6		16 14 22	(17) 4.2	10 15 10	(12) 2.9
-	5000	111 119 112	(114) 4.4	25 19 22	(22) 3.0	29 27 22	(26) 3.6		13 21 19	(18) 4.2	12 13 11	(12) 1.0
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		499 514 504	(506) 7.6	306 269 231	(269) 37.5	208 189 240	(212) 25.8		145 145 152	(147) 4.0	559 652 385	(532) 135.5

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Table 3              Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 08 June 2010						To: 11 June 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA-			TA98		TA1537
+	0	103 103 87	(98) 9.2#	14 14 13	(14) 0.6	26 30 36	(31) 5.0		24 25 31	(27) 3.8	9 14 14	(12) 2.9
+	50	104 110 99	(104) 5.5	13 7 10	(10) 3.0	25 30 31	(29) 3.2		21 18 23	(21) 2.5	10 15 14	(13) 2.6
+	150	119 100 93	(104) 13.5	12 13 14	(13) 1.0	30 34 32	(32) 2.0		23 25 25	(24) 1.2	13 10 7	(10) 3.0
+	500	90 84 92	(89) 4.2	14 14 15	(14) 0.6	30 24 26	(27) 3.1		18 24 29	(24) 5.5	15 11 11	(12) 2.3
+	1500	104 107 100	(104) 3.5	12 9 14	(12) 2.5	35 29 29	(31) 3.5		27 23 33	(28) 5.0	10 15 9	(11) 3.2
+	5000	86 98 84	(89) 7.6	14 14 12	(13) 1.2	30 30 27	(29) 1.7		24 23 32	(26) 4.9	12 9 14	(12) 2.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		405 389 381	(392) 12.2	219 197 229	(215) 16.4	300 323 324	(316) 13.6		202 133 128	(154) 41.4	242 273 216	(244) 28.5

 2AABP#

2AA    2-Aminoanthracene

BP      Benzo(a)pyren

#        Standard deviation

Table 4              Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 21 June 2010						To: 24 June 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA-			TA98		TA1537
-	0	122 107 101	(110) 10.8#	18 9 15	(14) 4.6	30 21 33	(28) 6.2		32 26 20	(26) 6.0	20 19 15	(18) 2.6
-	50	98 122 98	(106) 13.9	15 11 18	(15) 3.5	35 24 42	(34) 9.1		18 20 23	(20) 2.5	9 27 8	(15) 10.7
-	150	84 101 110	(98) 13.2	13 15 8	(12) 3.6	33 25 31	(30) 4.2		27 16 24	(22) 5.7	12 14 18	(15) 3.1
-	500	123 113 122	(119) 5.5	12 8 13	(11) 2.6	25 43 25	(31) 10.4		26 24 35	(28) 5.9	15 10 16	(14) 3.2
-	1500	112 106 97	(105) 7.5	11 13 15	(13) 2.0	33 37 33	(34) 2.3		31 20 23	(25) 5.7	11 14 14	(13) 1.7
-	5000	89 100 101	(97) 6.7	7 8 20	(12) 7.2	35 31 30	(32) 2.6		27 20 21	(23) 3.8	7 9 10	(9) 1.5
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		507 430 484	(474) 39.5	466 276 298	(347) 103.9	317 330 301	(316) 14.5		152 157 132	(147) 13.2	2206 2210 2107	(2174) 58.3

ENNG4NQO9AA#

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Table 5              Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 21 June 2010						To: 24 June 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA-			TA98		TA1537
+	0	102 107 118	(109) 8.2#	16 18 20	(18) 2.0	40 34 43	(39) 4.6		42 27 24	(31) 9.6	20 11 9	(13) 5.9
+	50	99 108 85	(97) 11.6	11 11 13	(12) 1.2	37 37 37	(37) 0.0		26 24 32	(27) 4.2	15 19 10	(15) 4.5
+	150	81 82 95	(86) 7.8	11 9 22	(14) 7.0	44 42 35	(40) 4.7		37 34 22	(31) 7.9	23 13 9	(15) 7.2
+	500	99 108 136	(114) 19.3	19 13 16	(16) 3.0	33 36 35	(35) 1.5		30 23 26	(26) 3.5	13 7 7	(9) 3.5
+	1500	101 76 75	(84) 14.7	14 9 15	(13) 3.2	32 41 34	(36) 4.7		25 25 30	(27) 2.9	10 11 10	(10) 0.6
+	5000	93 77 84	(85) 8.0	14 9 13	(12) 2.6	40 41 41	(41) 0.6		23 31 29	(28) 4.2	9 12 10	(10) 1.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		592 675 673	(647) 47.4	145 132 152	(143) 10.1	299 262 321	(294) 29.8		188 198 246	(211) 31.0	157 166 161	(161) 4.5

 2AABP#

2AA    2-Aminoanthracene

BP      Benzo(a)pyren

#        Standard deviation

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^-were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 16

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS
Preliminary Toxicity Test
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay were:


With (+) or without (-)
S9-mix Strain Dose (µg/plate)
0 0.15 0.5 1.5 5 15 50 150 500 1500 5000
- TA100 88 80 111 93 98 86 90 93 76 99 74
+ TA100 98 74 102 88 109 92 80 114 97 90 99
- WP2uvrA- 22 27 18 23 24 19 22 25 21 22 22
+ WP2uvrA- 19 32 30 31 22 23 25 24 22 20 23

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.
A history profile of vehicle and positive control values for 2008 and 2009 is presented in Appendix 2.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of theS9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table1              Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
96		20		22		16		7
95	(93)	21	(20)	18	(21)	21	(21)	12	(11)
88		18		22		25		13

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
89		12		53		18		10
96	(97)	18	(13)	34	(40)	21	(21)	13	(11)
107		10		32		23		11

Table 2              Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 08 June 2010						To: 11 June 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA-			TA98		TA1537
-	0	108 92 89	(96) 10.2#	22 29 25	(25) 3.5	23 32 27	(27) 4.5		22 15 27	(21) 6.0	16 14 10	(13) 3.1
-	50	86 99 115	(100) 14.5	26 27 27	(27) 0.6	33 29 27	(30) 3.1		14 21 14	(16) 4.0	10 15 16	(14) 3.2
-	150	107 111 97	(105) 7.2	20 25 24	(23) 2.6	24 30 24	(26) 3.5		20 18 15	(18) 2.5	15 16 13	(15) 1.5
-	500	98 103 102	(101) 2.6	29 21 23	(24) 4.2	32 26 30	(29) 3.1		20 19 18	(19) 1.0	12 7 15	(11) 4.0
-	1500	103 103 117	(108) 8.1	23 20 18	(20) 2.5	27 31 26	(28) 2.6		16 14 22	(17) 4.2	10 15 10	(12) 2.9
-	5000	111 119 112	(114) 4.4	25 19 22	(22) 3.0	29 27 22	(26) 3.6		13 21 19	(18) 4.2	12 13 11	(12) 1.0
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		499 514 504	(506) 7.6	306 269 231	(269) 37.5	208 189 240	(212) 25.8		145 145 152	(147) 4.0	559 652 385	(532) 135.5

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Table 3              Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 08 June 2010						To: 11 June 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA-			TA98		TA1537
+	0	103 103 87	(98) 9.2#	14 14 13	(14) 0.6	26 30 36	(31) 5.0		24 25 31	(27) 3.8	9 14 14	(12) 2.9
+	50	104 110 99	(104) 5.5	13 7 10	(10) 3.0	25 30 31	(29) 3.2		21 18 23	(21) 2.5	10 15 14	(13) 2.6
+	150	119 100 93	(104) 13.5	12 13 14	(13) 1.0	30 34 32	(32) 2.0		23 25 25	(24) 1.2	13 10 7	(10) 3.0
+	500	90 84 92	(89) 4.2	14 14 15	(14) 0.6	30 24 26	(27) 3.1		18 24 29	(24) 5.5	15 11 11	(12) 2.3
+	1500	104 107 100	(104) 3.5	12 9 14	(12) 2.5	35 29 29	(31) 3.5		27 23 33	(28) 5.0	10 15 9	(11) 3.2
+	5000	86 98 84	(89) 7.6	14 14 12	(13) 1.2	30 30 27	(29) 1.7		24 23 32	(26) 4.9	12 9 14	(12) 2.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		405 389 381	(392) 12.2	219 197 229	(215) 16.4	300 323 324	(316) 13.6		202 133 128	(154) 41.4	242 273 216	(244) 28.5

 2AABP#

2AA    2-Aminoanthracene

BP      Benzo(a)pyren

#        Standard deviation

Table 4              Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 21 June 2010						To: 24 June 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA-			TA98		TA1537
-	0	122 107 101	(110) 10.8#	18 9 15	(14) 4.6	30 21 33	(28) 6.2		32 26 20	(26) 6.0	20 19 15	(18) 2.6
-	50	98 122 98	(106) 13.9	15 11 18	(15) 3.5	35 24 42	(34) 9.1		18 20 23	(20) 2.5	9 27 8	(15) 10.7
-	150	84 101 110	(98) 13.2	13 15 8	(12) 3.6	33 25 31	(30) 4.2		27 16 24	(22) 5.7	12 14 18	(15) 3.1
-	500	123 113 122	(119) 5.5	12 8 13	(11) 2.6	25 43 25	(31) 10.4		26 24 35	(28) 5.9	15 10 16	(14) 3.2
-	1500	112 106 97	(105) 7.5	11 13 15	(13) 2.0	33 37 33	(34) 2.3		31 20 23	(25) 5.7	11 14 14	(13) 1.7
-	5000	89 100 101	(97) 6.7	7 8 20	(12) 7.2	35 31 30	(32) 2.6		27 20 21	(23) 3.8	7 9 10	(9) 1.5
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		507 430 484	(474) 39.5	466 276 298	(347) 103.9	317 330 301	(316) 14.5		152 157 132	(147) 13.2	2206 2210 2107	(2174) 58.3

ENNG4NQO9AA#

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Table 5              Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 21 June 2010						To: 24 June 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA-			TA98		TA1537
+	0	102 107 118	(109) 8.2#	16 18 20	(18) 2.0	40 34 43	(39) 4.6		42 27 24	(31) 9.6	20 11 9	(13) 5.9
+	50	99 108 85	(97) 11.6	11 11 13	(12) 1.2	37 37 37	(37) 0.0		26 24 32	(27) 4.2	15 19 10	(15) 4.5
+	150	81 82 95	(86) 7.8	11 9 22	(14) 7.0	44 42 35	(40) 4.7		37 34 22	(31) 7.9	23 13 9	(15) 7.2
+	500	99 108 136	(114) 19.3	19 13 16	(16) 3.0	33 36 35	(35) 1.5		30 23 26	(26) 3.5	13 7 7	(9) 3.5
+	1500	101 76 75	(84) 14.7	14 9 15	(13) 3.2	32 41 34	(36) 4.7		25 25 30	(27) 2.9	10 11 10	(10) 0.6
+	5000	93 77 84	(85) 8.0	14 9 13	(12) 2.6	40 41 41	(41) 0.6		23 31 29	(28) 4.2	9 12 10	(10) 1.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		592 675 673	(647) 47.4	145 132 152	(143) 10.1	299 262 321	(294) 29.8		188 198 246	(211) 31.0	157 166 161	(161) 4.5

 2AABP#

2AA    2-Aminoanthracene

BP      Benzo(a)pyren

#        Standard deviation

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^-were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 17

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

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

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 18

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
1. Both substances show low systemic toxicity in in vivo studies. A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure.
2. Substance similarities: Both salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.
Justification for no further testing for genetic toxicity: As sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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 PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolarity : No relevant changes in pH or osmolality of the test item formulations at concentrations of 3.16 to 2000 µg/mL medium were noted
- Water solubility: Soluble in water

RANGE-FINDING/SCREENING STUDIES: . In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Sodium dihydrogen orthophosphate/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium. Hence, 2000 µg/mL were employed as the top concentration for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Cytokinesis block proliferation index ranged between 1.26-1.48 for the test substance

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see tables below


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (micronucleus frequencies per 1000 cells):
Mitomycin C:
Mean: 46.9
SD: 35
range 17-137
Colchicine:
Mean: 25.9
SD: 9.5
range 15-63
cyclophosphamide:
Mean: 44.0
SD: 37.7
range 14-158
- Negative (solvent/vehicle) historical control data:

Without metabolic activation
Untreated control
mean 6.6
SD 2.9
range 2.0 - 17
95% Confidence interval 5.9 - 7.3

Vehicle control
mean 6.3
SD 3.1
range 2.0 - 18
95% Confidence interval 5.7 - 6.8

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI or RI in the case of the cytokinesis-block method

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

JUSTIFICATION FOR READ ACROSS; sodium and potassium orthophosphates.

The following substances are considered to be similar enough to facilitate read across for genetic toxicity endpoints:

Sodium dihydrogenorthophosphate, CAS: 7558-80-7

Disodium hydrogenorthophosphate, CAS: 7558-79-4

Trisodium orthophosphate, CAS: 7601-54-9

Potassium dihydrogenorthophosphate, CAS: 7778-77-0

Dipotassium hydrogenorthophosphate, CAS: 7758-11-4

Tripotassium orthophosphate, CAS: 7778-53-2

Potassium pentahydrogen bis(phosphate), CAS: 14887-42-4

All of the above substances have exhibited similar toxicity in acute oral and dermal studies and all are highly water soluble.

Read across is justified on the following basis:

1. Low systemic toxicity in in vivo studies.

A number of studies are provided to show that monovalent potassium and/or sodium inorganic orthophosphates exhibit low systemic toxicity via the oral route for both acute exposure and repeated dose exposure. These data are provided in Section 7.2 and Section 7.5 of the dossier for trisodium orthophosphate. The information provided in these records is considered to be indicative of a group of chemicals that are likely to behave in a similar way in vivo. 

2. Substance similarities:

All salts are monovalent inorganic phosphates, composed of a phosphate anion and an alkali metal cation. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.

 

Justification for no further testing for genetic toxicity:

 

Sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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.

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 and potassium orthophosphates and scientific justification for no further testing for the following reasons:

Sodium and potassium phosphates are routinely used in the nutrient broths that support bacterial colonies in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, sodium orthophosphates are also found in the metabolic activation mixture (e.g. S9-mix) which is used in an AMES 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.