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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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, tpyrophosphate will undergo hydrolysis (increased in acidic solutions) to form orthophosphate (PO43- anions).
3. Both substances have similar physicochemical and toxicological profiles. Differences arise in their local effects profile due to the increasing or decreasing acidity/alkalinity and buffering capacities of the substances. 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’.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
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.

Method

Species / strain
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
Controls
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.

Results and discussion

Test results
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.