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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 performed on an analogous substance is available for the Annex VIII Section 8.4.2 in vitro cytogenicity in mammalian cells and Section 8.4.3 in vitro gene mutation study in mammalian cells endpoint.

A range of supporting studies are available for all endpoints, including the Annex VIII Section 8.4.2 in vitro cytogenicity study in mammalian cells or in vitro micronucleus study.

None of the studies suggest the substance is mutagenic.

Link to relevant study records

Referenceopen allclose all

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

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

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

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

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

(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.

It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.


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

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

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose:
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
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 tissues of ICR random bred male mice, Sprague-Dawley adult male rats and Macaca mulatta adult male primates
Test concentrations with justification for top dose:
Plate test (S. typhimurium): 0.1%
Suspension tests (S. typhimurium): 1.13, 2,25%
Suspension tests (S. cerevisiae): 0.05, 0.1%
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Used in non-activation assays with tester strains TA 1535 and S. cerevisiae D4
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: quinacrine mustard
Remarks:
Used in a non-activation assay with tester strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Used in a non-activation assay with tester strain TA 1538
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: dimethylnitrosamine
Remarks:
Used in an activation assay with tester strain TA 1535 and S. cerevisiae D4
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Used in an activation assay with tester strains TA 1537 and TA 1538
Details on test system and experimental conditions:
Two types of assay were conducted; plate tests and suspension tests.

1. PLATE TESTS (bacteria only):

METHOD OF APPLICATION:
Nonactivation studies: plate incorporation
Activation studies: in suspension

DURATION
- Exposure duration: 4 days at 37°C

2. SUSPENSION TESTS (bacteria and yeast):

METHOD OF APPLICATION: in suspension

Nonactivation tests:

DURATION
- Exposure duration: 4 hours at 30°C (yeast assays), 1 hour at 37°C (bacterial assay)
- Selection time (if incubation with a selection agent): 48 hours at 37°C (bacterial assay), 3-5 days at 30°C

Activation tests:

DURATION
- Exposure duration: 4 hours at 30°C (yeast assays), 1 hour at 37°C (bacterial assay)


DETERMINATION OF CYTOTOXICITY
- Method: Each chemical was tested for survival against the specific indicator strains over a range of doses to determine 50% survival dose.
Evaluation criteria:
no data
Statistics:
No data
Species / strain:
other: TA 1353, TA 1357, TA 1358
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
other: Saccharomyces cerevisiae D4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test substance was soluble at the treatment concentration employed in this study. All tests were conducted in an aqueous environment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test date for toxicity determinations: September 15, 1974
The 50% survival level was determined for bacterial and yeast indicator organisms by conducting survival curves with the test compound at the following concentrations (w/w or v/v): For D4: 1, 2, 3, 4, 5. For S. typhimurium: 0.001, 0.005, 0.01, 0.05, 0.1%
-Concentrations of the test compound used in mutagenicity tests:

BACTERIA:
- Plate tests: 0.1%
-1/4 50% survival: 0.05%
-1/2 50% survival: 0.1%

YEAST:
- Plate tests: --
-1/4 50% survival: 1.13%
-1/2 50% survival: 2.25%
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

PLATE TESTS:

 

Table 2: Results of plate tests

 

TEST

SPECIES

TISSUE

REVERTANTS/PLATES

TA 1535

TA 1537

TA 1538

1

2

1

2

1

2

1. Nonactivation

 Positive control –(0.5 mL)

-

-

>103

>103

 

 

 

 

Positive control – QM (0.25 mg)

-

-

 

 

>102

>102

 

 

Positive control – NF (0.25 mg)

-

-

 

 

 

 

>102

>102

Solvent control - saline

 

 

2

1

2

4

 

 

Solvent control – DMSO (<10%)

 

 

 

 

 

 

5

1

Test compound – 1 (0.1%)

-

-

2

2

6

8

1

3

2. Activation

Positive control - DMNA

Mouse

Liver

>103

>103

 

 

 

 

Positive control – AAF

Liver

 

 

44

43

>102

>102

Positive control – DMNA

Lung

2

4

 

 

 

 

Positive control – AAF

Lung

 

 

9

3

13

8

Positive control – DMNA

Testes

1

5

 

 

 

 

Positive control – AAF

Testes

 

 

6

10

3

3

Solvent control –DMNA

-

3

0

 

 

 

 

Solvent control – AAF

-

 

 

10

5

1

0

Solvent control – saline

-

1

1

 

 

 

 

Solvent control –DMSO (<10%)

-

 

 

12

10

6

7

Test compound (0.1%)

Liver

2

0

7

9

17

12

Test compound (0.1%)

Lung

1

2

8

5

8

7

Test compound (0.1%)

Testes

0

0

9

4

12

15

Positive control – DMNA (25 µmoles)

Rat

Liver

>102

>102

 

 

 

 

Positive control – AAF (1.25 mg)

Liver

 

 

41

30

>102

>102

Positive control – DMNA (25 µmoles)

Lung

1

0

 

 

 

 

Positive control – AAF (1.25 mg)

Lung

 

 

7

10

5

0

Positive control – DMNA (25 µmoles)

Testes

3

0

 

 

 

 

Positive control – AAF (1.25 mg)

Testes

 

 

14

17

10

3

Positive control – DMNA (25 µmoles)

-

3

0

 

 

 

 

Positive control – AAF (1.25 mg)

-

 

 

10

5

1

0

Solvent control – saline

-

1

1

 

 

 

 

Solvent control –DMSO (<10%)

-

 

 

12

10

6

7

Test compound (0.1%)

Liver

1

0

9

4

18

5

Test compound (0.1%)

Lung

1

0

7

9

3

2

Test compound (0.1%)

Testes

0

2

4

11

5

6

Positive control – DMNA (25 µmoles)

Monkey

Liver

>102

>102

 

 

 

 

Positive control – AAF (1.25 mg)

Liver

 

 

32

46

>102

>102

Positive control – DMNA (25 µmoles)

Lung

0

4

 

 

 

 

Positive control – AAF (1.25 mg)

Lung

 

 

20

13

2

4

Positive control – DMNA (25 µmoles)

Testes

1

1

 

 

 

 

Positive control – AAF (1.25 mg)

Testes

 

 

10

11

3

7

Positive control – DMNA (25 µmoles)

-

3

0

 

 

 

 

Positive control – AAF (1.25 mg)

-

 

 

10

5

1

0

Solvent control – saline

-

1

1

 

 

 

 

Solvent control –DMSO (<10%)

-

 

 

12

10

6

7

Test compound

Liver

2

1

12

8

5

8

Test compound

Lung

1

0

7

7

8

5

Test compound

Testes

0

0

5

5

7

8

 

 

SUSPENSION TESTS:

 

Table 3. Results of suspension tests with and without activation

 

 

Test

Species

Organ

Salmonella reversion frequencies

D4 conversion frequencies (x 10-5)

TA 1535

TA 1537

TA 1538

Ade+

Trp+

Nonactivation

Positive control

-

-

1104.27

115.80

48.98

89.60

112.73

Negative control

-

-

1.46

11.81

10.61

6.55

4.37

High dose

-

-

2.12

10.29

7.39

6.44

4.93

Low dose

-

-

1.26

9.11

10.11

5.86

3.86

Activation

Solvent control

-

-

2.42

6.62

5.82

5.54

4.62

Chemical control

-

-

2.22

8.49

7.07

6.16

3.54

Positive control

Mouse

Liver

731.67

15.10

29.84

8.20

8.83

Positive control

Mouse

Lung

6.59

4.95

7.11

6.20

4.57

Positive control

Mouse

Testes

5.62

2.17

8.59

6.09

5.41

High dose

Mouse

Liver

0.97

11.07

7.29

7.17

6.05

Low dose

Liver

2.94

6.79

8.74

6.24

6.86

High dose

Lung

1.70

6.12

4.72

6.11

5.65

Low dose

Lung

1.00

4.82

8.00

7.71

7.13

High dose

Testes

0.69

3.07

5.78

7.19

4.04

Low dose

Testes

0.49

3.02

2.54

6.70

3.87

Solvent control

-

-

2.67

7.52

9.55

3.40

2.92

Chemical control

-

-

3.33

17.09

3.88

2.71

2.50

Positive control

Rat

Liver

245.70

29.39

42.80

9.42

10.27

Positive control

Rat

Lung

0.97

10.17

5.90

3.91

3.55

Positive control

Rat

Testes

1.68

8.83

6.08

0.23

2.02

High dose

Rat

Liver

1.33

9.83

6.91

--

3.01

Low dose

Liver

2.05

8.14

5.73

3.78

2.81

High dose

Lung

1.23

6.89

5.69

6.52

3.42

Low dose

Lung

0.71

6.94

7.69

5.72

2.63

High dose

Testes

1.49

6.94

1.80

6.68

4.20

Low dose

Testes

0.55

2.32*

3.47

4.40

2.45

Solvent control

-

-

2.49

16.19

13.85

2.86

1.33

Chemical control

-

-

4.27

18.27

9.18

4.06

1.43

Positive control

Monkey

Liver

206.49

26.72

30.32

9.81

2.82

Positive control

Monkey

Lung

2.49

8.64

13.79

3.94

3.62

Positive control

Monkey

Testes

2.79

6.91

8.71

7.10

3.84

High dose

Monkey

Liver

1.60

15.98

9.51

5.51

3.06

Low dose

Liver

1.97

11.72

9.09

5.41

5.84

High dose

Lung

1.68

14.89

11.45

2.56

3.84

Low dose

Lung

2.00

12.41

8.68

5.75

2.03

High dose

Testes

1.22

8.22

3.26

2.72

1.09

Low dose

Testes

3.81

5.79

3.56

5.00

2.50

 

* Data from repeat tests

 

Conclusions:
Tetrasodium pyrophosphate did not exhibit genetic activity in any of the assays employed and is considered to be non-mutagenic under the conditions of this study.
This data is submitted to provide a weight of evidence to support the conclusion that disodium dihydrogenpyrophosphate is unlikely to produce a positive genotoxic response.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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

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

(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.

It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.


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

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

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose:
read-across: supporting information
Qualifier:
no guideline followed
Principles of method if other than guideline:
The mutation test was carried out in accordance with the preincubation procedure described by Ames et al.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
other: TA 97 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.1, 0.5, 1, 5, 10 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Used with tester strain TA 97 without S9 mix Migrated to IUCLID6: (50 µg)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: no data

DETERMINATION OF CYTOTOXICITY
No data
Evaluation criteria:
No data
Statistics:
Kruskal-Wallis test and regression analysis
Species / strain:
other: TA 97 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Positive control data

Substance

Solvent

Dose

(µg/plate)

No. of revertants / plate (mean of 3 plates)

TA 97

TA 102

-S9

+S9

-S9

+S9

9-Aminoacridine

DMSO

50 µg

627 ± 2131

 

 

 

Mitomycin C

DMSO

0.5 µg

 

 

2438 ± 236

 

2-Aminoanthracene

DMSO

5 µg

 

2104 ± 656

 

1144 ± 226

1. mean and standard deviation (n=19)

Table 2: Results of an AMES study conducted on sodium pyrophosphate:

Substance

Solvent

Dose

(mg/plate)

No. of revertant / plate (mean of 3 plates)

TA 97

TA 102

-S9

+S9

-S9

+S9

Sodium pyrophosphate

DW

10

122

187

334

328

5

125

195

348

333

1

114

205

334

489

0.5

130

207

342

476

0.1

150

202

341

500

0

151

184

340

496

Conclusions:
The authors conclude that sodium pyrophosphate is non-mutagenic under the conditions of this study.
This data is submitted to provide a weight of evidence to support the conclusion that disodium dihydrogenpyrophosphate is unlikely to produce a positive genotoxic response.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No postive control data
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Remarks:
Study pre-dates GLP
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
other: TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with
Metabolic activation system:
Rat liver S-9
Test concentrations with justification for top dose:
Maximum dose: 10.0 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Phosphate buffer
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
exposed to appropriate solvent or untreated
True negative controls:
not specified
Positive controls:
no
Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 2 days at 37 °C
Evaluation criteria:

The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). A negative result indicates that no significant increases in the numbers of revertant colonies were detected in any S. typhimurium strains at the maximum dose.
Statistics:
No data
Species / strain:
other: TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not examined
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
No data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Results as presented in study:

Additive

Max dose

(mg/plate)

Result

Disodium dihydrogen phosphate

10.0

Negative

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

This data is submitted to provide a weight of evidence to support the conclusion that disodium dihydrogenpyrophosphate is unlikely to produce a positive genotoxic response.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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

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

(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.

It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.


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

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

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose:
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:
Stduy 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:
not specified
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:
not specified
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: The substance was tested over a series of concentrations. The dose range employed in the study was below a concentration that demonstrated any toxic effect.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 2. Results of anstudy on MCTR-257

Test

Dosage

(μL)

Revertants per plate

TA-1353

TA-1357

TA-1358

TA-98

TA-100

D4*

1

2

1

2

1

2

1

2

1

2

1

2

Non-activation

Solvent control

-

11

 

10

 

11

 

30

 

154

 

68

 

Positive control

**

236

 

901

 

408

 

721

 

530

 

>1000

 

Test compound

0.001

15

 

5

 

11

 

20

 

191

 

48

 

0.01

8

 

13

 

8

 

24

 

203

 

55

 

0.1

25

 

6

 

5

 

19

 

212

 

54

 

1.0

19

 

9

 

5

 

21

 

198

 

44

 

5.0

12

 

10

 

5

 

20

 

210

 

39

 

Activation

Solvent control

-

13

 

16

 

16

 

43

 

124

 

35

 

Positive control

***

242

 

208

 

428

 

650

 

>1000

 

82

 

Test compound

0.001

17

 

23

 

20

 

45

 

162

 

50

 

0.01

19

 

21

 

22

 

41

 

170

 

61

 

0.1

16

 

15

 

23

 

48

 

170

 

58

 

1.0

14

 

23

 

18

 

40

 

154

 

52

 

5.0

16

 

6

 

21

 

39

 

179

 

52

 

* TRY+ convertants per plate.

**

TA-1535

MNNG

10 μg/plate

TA-1537

QM

10 μg/plate

TA-1538

NF

100 μg/plate

TA-98

NF

100 μg/plate

TA-100

MNNG

10 μg/plate

D4

MNNG

10 μg/plate

***

TA-1535

ANTH

100 μg/plate

TA-1537

AMQ

100 μg/plate

TA-1538

AAF

100 μg/plate

TA-98

AAF

100 μg/plate

TA-100

ANTH

100 μg/plate

D4

DMNA

100 micromoles/plate

Conclusions:
The test compound MCTR-257 did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered not-mutagenic under these test conditions.
This data is submitted to provide a weight of evidence to support the conclusion that disodium dihydrogenpyrophosphate is unlikely to produce a positive genotoxic response.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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

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

(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.

It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.


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

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

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Remarks:
Study published in the literature and therefore GLP compliance not specified.
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9: induced with Aroclor 1254 or a combination of phenobarbitone and β-naphthoflavone.
Test concentrations with justification for top dose:
0, 301.25, 602.5, 1,205, 2,410, 4,820 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: No data
Untreated negative controls:
yes
Remarks:
0 µg/plate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Remarks:
0 µg/plate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Remarks:
0 µg/plate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (2-AF)
Untreated negative controls:
yes
Remarks:
0 µg/plate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
Migrated to IUCLID6: (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); without metabolic activation
preincubation; with metabolic activation

DURATION
- Preincubation period: 20 minutes or more
- Exposure duration: 48-72 hrs at 37°C


NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity detected by a reduction in the number of reverting colonies, a clearing or diminution of the background lawn, or the degree of survival of treated cultures.
Evaluation criteria:
Number of revertant colonies per plate were counted. The mutant frequency was expressed as the quotient of the number of revertant colonies over the number of colonies in the negative control. A mutagenic potential of a test item was assumed if the mutant frequency is 2.0 or higher.
Statistics:
No data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Tetrasodium pyrophosphate was found to be non-genotoxic in an AMES test both with and without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

No classification for in vitro genetic toxicity is proposed. This is based on a weight of evidence approach using all relevant data on sodium acid pyrophosphate and its analogues. No further testing is considered necessary for the following reasons:

Sodium and potassium phosphates are routinely used in the nutrient broths that support cell cultures in the laboratory and as such bacteria are constantly exposed to these inorganic phosphates. In addition, disodium dihydrogenpyrophosphate will hydrolyse to sodium orthophosphates which are also found in the metabolic activation mixture (e.g. S9-mix) which is used in a chromosomal aberration test to determine whether a test chemical can be metabolized within the body to produce a compound that may be genotoxic. The constant exposure of bacteria to these materials suggests that they pose no inherent risk of genotoxicity.

As such and in accordance with Regulation (EC) No.1272/2008 (EU CLP) no classification is proposed.