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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 07 September 2016 to 21 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

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

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

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

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

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Cross-reference
Reason / purpose:
read-across: supporting information

Data source

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

Materials and methods

Test guidelineopen allclose all
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

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: 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:

Method

Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.