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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

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
read-across: supporting information
Qualifier:
equivalent or similar to
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:
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:
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:
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:
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 controla

-

-

>103

>103

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 controlb

Mouse

Liver

>103

>103

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

>103

>103

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

>103

>103

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.
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:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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:
read-across: supporting information
Reason / purpose:
read-across source
Species / strain:
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:
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 controla

-

-

>103

>103

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 controlb

Mouse

Liver

>103

>103

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

>103

>103

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

>103

>103

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:
Disodium phosphate is estimated to be non-mutagenic under the conditions of this study.
Executive summary:

Disodium 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 disodium hydrogen 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

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
read-across: supporting information
Qualifier:
equivalent or similar to
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:
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:
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:
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 controla

-

-

>103

>103

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 controlb

Mouse

Liver

>103

>103

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

>103

>103

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.
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:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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:
read-across: supporting information
Reason / purpose:
read-across source
Species / strain:
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:
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 controla

-

-

>103

>103

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 controlb

Mouse

Liver

>103

>103

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

>103

>103

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

Executive summary:

Disodium phosphate was estimated to be non-mutagenic as found in the source study performed with monosodium phosphate anhydrous. As explained in the justification for type of information, the differences in molecular structure between disodium hydrogen 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

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
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

POS1

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

POS1

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

POS1

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

POS1

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

POS1

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:
negative with metabolic activation
negative without metabolic activation

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.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
read-across: supporting information
Qualifier:
equivalent or similar to
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:
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:
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.
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:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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:
read-across: supporting information
Reason / purpose:
read-across source
Species / strain:
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:
Disodium phosphate is estimated to be non-mutagenic under the conditions of this study.
Executive summary:

Disodium 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 disodium hydrogen 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

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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:
read-across: supporting information
Qualifier:
equivalent or similar to
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:
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:
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:
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:
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.
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:
weight of evidence
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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:
read-across: supporting information
Reason / purpose:
read-across source
Species / strain:
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:
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:
Disodium phosphate is estimated to be non-mutagenic under the conditions of this study.
Executive summary:

Disodium 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 disodium hydrogen 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

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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:
read-across: supporting information
Qualifier:
equivalent or similar to
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:
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:
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:
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:
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.
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:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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:
read-across: supporting information
Reason / purpose:
read-across source
Species / strain:
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:
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:
Disodium phosphate is estimated to be non-mutagenic under the conditions of this study.
Executive summary:

Disodium 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 disodium hydrogen 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

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
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:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
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. certificate)
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.

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:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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:
read-across: supporting information
Reason / purpose:
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.

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:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted September 26, 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 chi2 -test
                            Highly purified water (vehicle control)
 1 345  144  11  1.33  100  1000 
 9 330  162  1.36  100  1000 
                            Sodium dihydrogen orthophosphate
 6 125  340  155  1.33  100  1000  n.s. 
 14 125  320  169  11  1.38  106  1000  n.s. 
 5 250  342  146  12  1.34  103  1000  n.s. 
 13 250  296  181  23  1.45  125  1000  n.s. 
 4 500  246  237  17  1.54  164  1000  n.s. 
 12 500  306  183  11  1.41  114 1000  n.s. 
 3 1000  344  143  13  1.34  103  1000  n.s. 
 11 1000  310  177  13  1.41  114  1000  n.s. 
 2 2000  334  160  1.34  103  1000  n.s. 
 10 2000  320  172  1.38  106  1000  n.s. 
            Mitmycin C                
 7 0.2  309  183  1.40  121  1000  36  s. 
 15 0.2  326  171  135  97  1000  39  s. 

n.s. = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI = Replicative test

Table 4: Experiment without metabolic activation (S9 mix) - 24hr exposure

 Culture number Concentration [µg/mL medium]  mononucleate  Number of binucleate cells#  multinucleate  CBPI  RI [%]  Number of binucleate cells scored  Number of micronucleated cells per 1000 binucleate cells  Significane chi2-test
                            Highly ourified water (vehicle control)
 1 354  134  12  1.32  100  1000 
 9 378  106  16  1.28  100  1000 
                            Sodium dihydrogen orthophosphate
 5 250  366  105  29  1.33  103  1000  n.s. 
 13 250  379  120  1.24  86  1000  n.s. 
 4 500  363  122  15  1.30  94  1000  n.s.             
 12 500  356  130  14  1.32  114  1000  n.s. 
 3 1000  377  110  13  1.27  84  1000  n.s. 
 11 1000  382  112  1.25  89  1000  n.s. 
 2 2000  370  119  11  1.28  88  1000  n.s. 
 10 2000  389  103  1.24  86  1000  n.s. 
     Colchicine                       
 7 0.02  403  86  11  1.22  69  1000  61  s. 
 15 0.02  408  85  1.20  71  1000  46  s. 

n.s = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI = Replicative Index

Table 5: Experiment with metabolic activation (+S9 mix) -4hr exposure

 Culture number Concentration [µg/mL medium]  mononucleate  Number of binucleate cells#  multinucleate  CBPI  RI [%]  Number of binucleate cells scored  Number of micronucleated cells per 1000 binucleate cells  Significance chi2-test 
                Highly purified water (vehicle control)            
 1 366  128  1.28  100  1000 
 9 343  143  14  1.34  100  1000 
                            Sodium dihydrogen orthphosphate
 6 125  320  165  15  1.39  139  1000  n.s. 
 14 125  345  146  1.33  97  1000  n.s. 
 5 250  367  127  6 1.28  100  1000  n.s. 
 13 250  362  127  11  1.30  88  1000  n.s 
 4 500  344  144  12  1.34  121  1000  n.s. 
 12 500  288  103  1.24  71  1000  n.s. 
 3 1000  325  163 12 1.37  132  1000  n.s. 
 11 1000  380  116  1.25  74  1000  n.s. 
 2 2000  378  106  16  1.28  100  1000  n.s. 
 10 2000  361  130  1.30  88  1000  n.s. 
                            Cyclophosphamide
 7 20  367  127  1.28  100  1000  26  s. 
 15 20  362  132  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:
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:
read-across: supporting information
Reason / purpose:
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 chi2 -test
                            Highly purified water (vehicle control)
 1 345  144  11  1.33  100  1000 
 9 330  162  1.36  100  1000 
                            Sodium dihydrogen orthophosphate
 6 125  340  155  1.33  100  1000  n.s. 
 14 125  320  169  11  1.38  106  1000  n.s. 
 5 250  342  146  12  1.34  103  1000  n.s. 
 13 250  296  181  23  1.45  125  1000  n.s. 
 4 500  246  237  17  1.54  164  1000  n.s. 
 12 500  306  183  11  1.41  114 1000  n.s. 
 3 1000  344  143  13  1.34  103  1000  n.s. 
 11 1000  310  177  13  1.41  114  1000  n.s. 
 2 2000  334  160  1.34  103  1000  n.s. 
 10 2000  320  172  1.38  106  1000  n.s. 
            Mitmycin C                
 7 0.2  309  183  1.40  121  1000  36  s. 
 15 0.2  326  171  135  97  1000  39  s. 

n.s. = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI = Replicative test

Table 4: Experiment without metabolic activation (S9 mix) - 24hr exposure

 Culture number Concentration [µg/mL medium]  mononucleate  Number of binucleate cells#  multinucleate  CBPI  RI [%]  Number of binucleate cells scored  Number of micronucleated cells per 1000 binucleate cells  Significane chi2-test
                            Highly ourified water (vehicle control)
 1 354  134  12  1.32  100  1000 
 9 378  106  16  1.28  100  1000 
                            Sodium dihydrogen orthophosphate
 5 250  366  105  29  1.33  103  1000  n.s. 
 13 250  379  120  1.24  86  1000  n.s. 
 4 500  363  122  15  1.30  94  1000  n.s.             
 12 500  356  130  14  1.32  114  1000  n.s. 
 3 1000  377  110  13  1.27  84  1000  n.s. 
 11 1000  382  112  1.25  89  1000  n.s. 
 2 2000  370  119  11  1.28  88  1000  n.s. 
 10 2000  389  103  1.24  86  1000  n.s. 
     Colchicine                       
 7 0.02  403  86  11  1.22  69  1000  61  s. 
 15 0.02  408  85  1.20  71  1000  46  s. 

n.s = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI = Replicative Index

Table 5: Experiment with metabolic activation (+S9 mix) -4hr exposure

 Culture number Concentration [µg/mL medium]  mononucleate  Number of binucleate cells#  multinucleate  CBPI  RI [%]  Number of binucleate cells scored  Number of micronucleated cells per 1000 binucleate cells  Significance chi2-test 
                Highly purified water (vehicle control)            
 1 366  128  1.28  100  1000 
 9 343  143  14  1.34  100  1000 
                            Sodium dihydrogen orthphosphate
 6 125  320  165  15  1.39  139  1000  n.s. 
 14 125  345  146  1.33  97  1000  n.s. 
 5 250  367  127  6 1.28  100  1000  n.s. 
 13 250  362  127  11  1.30  88  1000  n.s 
 4 500  344  144  12  1.34  121  1000  n.s. 
 12 500  288  103  1.24  71  1000  n.s. 
 3 1000  325  163 12 1.37  132  1000  n.s. 
 11 1000  380  116  1.25  74  1000  n.s. 
 2 2000  378  106  16  1.28  100  1000  n.s. 
 10 2000  361  130  1.30  88  1000  n.s. 
                            Cyclophosphamide
 7 20  367  127  1.28  100  1000  26  s. 
 15 20  362  132  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:
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:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of Inspection: 16-19 April 2013 Date of Signature on Certificate: 14 May 2014
Type of assay:
other: gene mutation in mammalian cells
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No: 1541481
- Expiration date of the lot/batch: November 2018
- Purity test date: 99.8 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in a tightly closed container and stored at a dry, cool and well-ventilated place

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Completely dissolved in highly purified water

FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution

OTHER SPECIFICS:
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The indicator cell used for this study was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-). The particular clone (3.7.2C) used in this assay is isolated by ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA.
- Methods for maintenance in cell culture if applicable: Master stock cultures were maintained in liquid nitrogen.Laboratory cultures were periodically checked for karyotype stability and the absence of mycoplasma contamination by culturing methods. To reduce the background mutant frequency (spontaneous frequency) of TK / mutants to a level as low as possible, cell cultures were exposed to conditions that select against the TK / phenotype (exposure to aminopterin or methotrexate).


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cells used during the experimental studies were maintained in growth medium RPMI 1640 with glutamaxTM medium supplemented with Pluronic® F68 , gentamycin , amphotericin B1 and horse serum1 (10% by volume).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver enzymes (S9 fraction) and an energy producing system comprising nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and glucose-6-phosphate.
Test concentrations with justification for top dose:
Based on the results of the preliminary study five concentrations of 125, 250, 500, 1000 and 2000 µg for the experiments without and with metabolic activation were employed in the mutagenicity tests.

A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation. A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000, and 2000 µg PRAYPHOS™ MSP FG/FG GR/mL medium were tested for cytotoxicity. Cytotoxicity (decreased survival) was noted at a concentration of 1000 or 2000 µg/mL in the presence of metabolic activation (3-hour exposure). No changes in pH or osmolality were noted in the test item formulations compared to the control.
Hence, in the main study the highest concentration employed was 2000 µg PRAYPHOS™ MSP FG/FG GR/mL medium in the experiments without and with metabolic activation.


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (purified water) treatment groups were used as the negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle treatment groups (purified water) were used as the negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (490) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at five dose levels, in duplicate, together with vehicle (water) and positive controls. The exposure groups used were as follows: 3 hour exposures both with and without metabolic activation. A repeat experiment was used with 3 hour exposure for the metabolic activation groups and 24 hours for the without metabolic activation groups.

The dose range of test material was selected following the results of a preliminary toxicity test and was 125 to 2000 µg/ml for all exposure groups.

In the preliminary study, cytotoxicity (decreased survival) was noted at a concentration of 1000 or 2000 µg/mL in the presence of metabolic activation (3-hour exposure). Hence, in the main study the highest concentration employed was 2000 µg PRAYPHOS™ MSP FG/FG GR/mL medium in the experiments without and with metabolic activation. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the exposure groups.
Evaluation criteria:
Please see Interpretation of Results in "Any other information and methods incl. tables" section. As this section will not accommodate the required information.
Statistics:
Please see Interpretation of Results in "Any other information and methods incl. tables" section. As this section will not accommodate the required information.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In preliminary experiment, cytotoxicity was noted at a concentration of 1000 or 2000 ug/mL in the presence of metabolic activation (3-hour exposure)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Results
A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation.A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000, and 2000 µg sodium dihydrogenorthophosphate/mL medium were tested for cytotoxicity. Cytotoxicity (decreased survival) was noted at a concentration of 1000 or 2000 µg/mL in the presence of metabolic activation (3-hour exposure). For details, seeTable 1. No changes in pH or osmolality were noted in the test item formulations compared to the control (seeText table 7-1 'pH values and osmolality').

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

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

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

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

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

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

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

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

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

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

Please see Attached Tables

Due to the nature and quantity of the tables it was not possible to insert them in this section.

Text table 7-1 pH values and osmolality

 

Concentration of

pH value

Osmolality

Sodium dihydrogenorthophosphate

 

[mOsmol/kg]

[µg/mL medium]

 

 

Medium

7.59

290.0

Negative control

7.60

290.0

10

7.62

285.0

31.6

7.65

285.0

100

7.53

285.0

316

7.67

285.0

1000

6.96

290.0

2000

6.68

310.0

Conclusions:
Under the present test conditions, sodium dihydrogenorthophosphate, tested up to a concentration of 2000 µg/mL medium, the recommended maximum concentration by the guideline, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions, the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, sodium dihydrogenorthophosphate also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that sodium dihydrogenorthophosphate, tested up to a concentration of 2000 µg/mL medium, neither induced mutations nor had any chromosomal aberration potential.
Executive summary:

In order to investigate the mutagenic potential on mammalian cells, the sodium dihydrogenorthophosphatewas assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/‑) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254‑induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours, the experiment with S9 mix was carried out in two independent assays.

Sodium dihydrogenorthophosphate was completely dissolved in highly purified water. The vehicle highly purified water served as the negative control.

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

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

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

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

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

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

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

The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3‑MC) caused pronounced increases in the mutation frequency of 474.25 and 652.55 per 106clonable cells in the case of MMS and of 387.42 and 489.98 per 106clonable cells in the case of 3-MC. As the increase in the small colony mutation frequency was at least 150 x 10-6above the concurrent negative control, anabsolute increase in totalmutation frequency was at least 300 x 10-6 for the positive controls and the mean relative total growth (RTG) greater than or equal to 10%, the acceptance criteria for the positive controls were met.

Endpoint:
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:
read-across source
Reason / purpose:
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:
Disodium 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:

Disodium 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 disodium 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.
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 disodium hydrogenorthophosphate. 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 PO43-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 PO43-ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems.