Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria: An adaptation is submitted.

In vitro cytogenicity study: A key study on an analogous substance is submitted. The study is performed in accordance with OECD 487 and under the conditions of GLP.

In vitro gene mutation in mammalian cells: A key study on an analogous substance is submitted. The study is performed in accordance with OECD 490 and under the conditions of GLP.

Link to relevant study records

Referenceopen allclose all

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
In this instance, the substance similarities are as follows:
1. The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table; sodium and phosphate anion(s).
2. Both substances will ultimately dissociate into the common breakdown products; Na+ cations and PO43- anions. In biological systems, the metaphosphate will undergo hydrolysis (increased in acidic solutions) to form shorter phosphate chains and ultimately phosphate (PO43- anions).
3. Both substances have similar physicochemical and toxicological profiles. The ionic components of both the source and the target compounds are essential micronutrients and their uptake is tightly regulated.

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
In this instance, the substance similarities are as follows:
1. The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table; sodium and phosphate anion(s).
2. Both substances will ultimately dissociate into the common breakdown products; Na+ cations and PO43- anions. In biological systems, the metaphosphate will undergo hydrolysis (increased in acidic solutions) to form shorter phosphate chains and ultimately phosphate (PO43- anions).
3. Both substances have similar physicochemical and toxicological profiles. The ionic components of both the source and the target compounds are essential micronutrients and their uptake is tightly regulated.

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:
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. Sodium metaphosphate is not considered to be clastogenic.
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
In this instance, the substance similarities are as follows:
1. The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table; sodium and phosphate anion(s).
2. Both substances will ultimately dissociate into the common breakdown products; Na+ cations and PO43- anions. In biological systems, the metaphosphate will undergo hydrolysis (increased in acidic solutions) to form shorter phosphate chains and ultimately phosphate (PO43- anions).
3. Both substances have similar physicochemical and toxicological profiles. The ionic components of both the source and the target compounds are essential micronutrients and their uptake is tightly regulated.

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
In this instance, the substance similarities are as follows:
1. The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table; sodium and phosphate anion(s).
2. Both substances will ultimately dissociate into the common breakdown products; Na+ cations and PO43- anions. In biological systems, the metaphosphate will undergo hydrolysis (increased in acidic solutions) to form shorter phosphate chains and ultimately phosphate (PO43- anions).
3. Both substances have similar physicochemical and toxicological profiles. The ionic components of both the source and the target compounds are essential micronutrients and their uptake is tightly regulated.

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:
Sodium metaphosphate 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:

Sodium metaphosphate 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 sodium metaphosphate 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

Short description of key information:
Supporting data on the ability of sodium metaphosphate to induce reverse mutations in bacterial colonies (in the form of AMES studies) are available. However, as the data are either lacking in methodological detail or do not meet important criteria of today's standard methods (in particular neither studies look at the ability of sodium metaphosphate to induce cross-linking mutations) neither can be considered as a key study for this endpoint. Further generation of data is not considered to be scientifically justified for the following reasons:

The hydrolysis products of sodium metaphosphate (sodium and orthophosphate ions) are routinely used in the nutrient broths that support cell or bacterial colonies in the laboratory and as such cells 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 many in vitro genotoxicity studies to determine whether a test chemical can be metabolized within the body to produce a compound that may or may not be genotoxic. The constant exposure of bacteria and cells to these materials suggests that they pose no inherent risk of genotoxicity.
In addition, the Na+ and PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems. As the ultimate breakdown product of metaphosphate is known to be orthophosphate.

Justification for classification or non-classification

No classification for genotoxicity is proposed for sodium metaphosphate, in accordance with Regulation (EC) No. 1272/2008 for the reasons detailed in the endpoint adaptations. No further testing is considered necessary.