Phosphonic acid, diammonium salt, monohydrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 30 October to 18 march 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study, well conducted on the similar substance Reaction mass of dipotassium phosphonate and Phosphonic acid, potassium salt (1:1). Rationale for Read Across is attached at point 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (in Vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Potassium Phosphonate KH2PO3/K2HPO3

IUPAC Name:

Potassium Phosphonate KH2PO3/K2HPO3

Test material form:

other: liquid

Details on test material:

- Name of test material: potassium phosphonate KH2PO3/K2HPO3
- Substance type: inorganic
- Physical state: limpid fluid colourless solution
- Storage condition of test material: room temperature
- Stability: no assay of test item stability, nor its concentration and homogeneity in solvent were undertaken
- Expiration date of the lot/batch: 16/12/2013
- Purity: 50%
- Preparation:immediately before use in sterile water of injectable grade on a weight/volume basis, with correction for the displacement due to the volume of the test item. All solutions were used within 25 minutes from the initial formulation. Treatments were performed under safe light conditions when the positive control Colchicine was included.
The solubility of the test item was assess during the test: during the first main experiment, the test item was found to be miscible with sterile distilled water of injectable grade. Following treatment with the test item, no opacity of the medium nor precipitation of the test item was observed at the beginning or end of treatment, both in the absence or presence of S9 metabolism.
The osmolality and the pH were assess during the test.
Following treatment with the test item, no remarkable variation of osmolality and pH was observed in the absence or presence of S9 metabolism in any experiment.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Source: Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK
This cell line, V79 4(H) can be traced back directly to the original V79 isolate prepared by Ford and Yerganian (1958). The karyotype, generation time and plating efficiency have been checked. The cells are checked at regular intervals for the absence of mycoplasmal contamination.
- Storage: in liquid nitrogen and subcultures are prepared from the frozen stocks for experimental use

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

S9 time (hours)         Dose levels µg/mL.
1             -              3             5000, 2500, 1250, 625 and 313
1             +             3             5000, 2500, 1250, 625 and 313
2             -              24          5000, 3570, 2550, 1820, 1300 and 930
2             +             3             5000, 3570, 2550, 1820, 1300 and 930

Vehicle / solvent:

sterile injectable water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

sterile injectable water grade

Positive controls:

yes

Positive control substance:

mitomycin C

other: Colchicine

Remarks:

Absence of S9 metabolism

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

sterile injectable water grade

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Presence of S9 metabolism

Details on test system and experimental conditions:

S9 TISSUE HOMOGENATE
- Source: Trinova Biochem GmbH (producer: MOLTOX, Molecular Toxicology, Inc., USA)
- Species: rat
- Strain: Sprague Dawley
- Sex: male
- Tissue: liver
- Inducing Agents: Phenobarbital-5,6-Benzoflavone
S9 MIX (15 ml)
S9 tissue fraction, NADP, G-6-P, Hepes buffer, KCl, HBSS Hank's Balanced salt solution
CULTURE MEDIUM
EMEM Minimal medium, EMEM Complete, DMEM Minimal medium, DMEM Complete medium
EXPERIMENTAL DESIGN
- Preliminary toxicity assay;
- Treatment:
ASSAY n.1= without S9, for 3 hours, dose levels (5000-2500-1250-625-313, 156, 78.1, 39.1 µg/mL)
ASSAY n.1= with S9, for 3 hours, dose levels (5000-2500-1250-625-313, 156, 78.1, 39.1 µg/mL)
ASSAY n.2= final concentration of 3 µg/mL, incubated for 26 hours at 37 °C. until harvesting.
Harvesting and slide preparation/evaluation
DEVIATION FROM STUDY PROTOCOL
Procedures were in agreement with the Study Protocol, with the exception of the following deviations:
- for the first main experiment, scoring of binucleated cells and not mononucleated cells was performed for cultures treated with Colchicine.
- due to technical reasons, DMEM complete medium and not EMEM complete medium was used for the first main experiment.
Those deviations were not considered to have affected the integrity or purpose of the study.

Evaluation criteria:

IDENTIFICATION CRITERIA
- The micronucleus diameter was less than 1/3 of the nucleus diameter;
- The micronucleus diameter was greater than 1/16 of the nucleus diameter;
- No overlapping with the nucleus was observed;
- The aspect was the same as the chromatin;
CRITERION FOR OUTCOME
- Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations;
- The proportion of micronucleated cells at such data points exceeds the normal range. If the increases fall within the range of values normally observed in the negative control cultures, the test item cannot be classified as positive. Any significant increases over the concurrent negative controls were therefore compared with historical control values derived from recent studies;
- There is a significant dose-relationship.

Statistics:

A modified chi-squared test is used to compare the number of cells bearing micronuclei in control and treated cultures.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

EVALUATION OF RESULTS
Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the negative control value was observed at any dose level in the first main experiment.
In the second main experiment, statistically significant increases in the incidence of micronucleated cells over the concurrent negative control value were observed at the high and intermediate dose levels selected for scoring.
Besides, a dose-effect relationship was indicated and at the highest dose level, incidences exceeded the range of RTC historical values for negative controls in both replicate cultures.
Statistically significant increases in the proportion of micronucleated cells compared with the relevant control values were observed in the cultures treated with the positive controls Cyclophosphamide, Mitomycin-C and Colchicine, indicating the correct functioning of the assay system.
SELECTION OF DOSE LEVELS FOR SCORING
For the first main experiment, following treatment with the test item, no cytotoxicity was observed in the absence or presence of S9 metabolism at any dose level.
For the second main experiments, moderate cytotoxicity (36%) was observed at the highest dose level selected for treatment (4000 µg/mL), mild cytotoxicity was observed at the two lower dose levels of 3330 and 2780 g/mL (22% and 20%, respectively). No cytotoxicity was observed over the remaining dose range.
The highest dose level for genotoxicity assessment is selected as a dose which produces a substantial cytotoxicity compared with the solvent control. Ideally the cytotoxicity should be between 50% and 60%. In the absence of cytotoxicity the highest treatment level is selected as the highest dose level for scoring. Two lower dose levels are also selected for the scoring of micronuclei.
The summary data are shown in table 1 and 2.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Summary table of the treatment time 3h, sampling time 27 hours (main assay 1)

		Presence of S9 metabolism			Absence of S9 metabolism
Treatment	Dose level (mg/mL)
		%Mn cells	Sig.	%Cytotoxicity	%Mn cells	Sig.	%Cytotoxicity
Untreated	0.0	1.30		2	0.85		3
Solvent 10%	0.0	0.90		0	0.70		0
Test item	1250	1.25	NS	-4	0.85	NS	2
Test item	2500	0.80	NS	-1	0.90	NS	5
Test item	5000	0.60	NS	-6	0.85	NS	1
Mitomycin-C	0.300	-		-	4.15	***	12
Colchicine	2.50	-		-	2.50	***	-6
Cyclophosphamide	10.0	4.10	***	58	-		-

Table 2. Summary table of the treatment time 26h, sampling time 27 hours (main assay 2)

		Absence of S9 metabolism
Treatment	Dose level (mg/mL)
		%Mn cells	Sig.	%Cytotoxicity
Untreated	0.0	0.45		1
Solvent 10%	0.0	0.55		0
Test item	2780	0.90	NS	20
Test item	3300	1.40	*	22
Test item	4000	4.50	***	36
Mitomycin-C	0.100	6.35	***	32
Colchicine	0.250	12.2	***	98

%Mn cells: Percentage of cells bearing micronuclei

Sig.: Statistical significance

NS: Not significant

-: Not tested or not selected for scoring

*: Statistically significant at p<0.05

** : Statistically significant at p<0.01

***: Statistically significant at p<0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation
negative with metabolic activation

It can be concluded that Potassium phosphonate – Multicomponent induces micronuclei in Chinese hamster V79 cells after in vitro treatment in the absence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

Potassium Phosphonate multicomponent (K2HPO3/KH2PO3) was assayed for the ability to induce micronuclei in Chinese hamster V79 cells, following in vitro treatment in the presence and absence of S9 metabolic activation.

Two main experiments were performed.

In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 27 hours corresponding to approximately 2.0 cell cycle lengths was used.

As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 26 hours was used.

For the first main experiment, the maximum dose level for treatment was selected in agreement with the Study Protocol.

Dose levels of 5000, 2500, 1250, 625, 313, 156, 78.1 39.1 µg/mL were used both in the absence and presence of S9 metabolism.

Based on the cytotoxicity observed in the first experiment, dose levels of 4000, 3300, 2780, 2310, 1930, 1610, 1340, 1120, 930 and 775 µg/mL were selected for the second main experiment.

Test item solutions were prepared in sterile water for injection.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin-B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels were selected for the scoring of micronucleated cells on the basis of the cytotoxicity of the test item treatments calculated by thecytokinesis-block proliferation index (CBPI).

In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 27 hours corresponding to approximately 2.0 cell cycle lengths was used.

As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 26 hours was used.

For the first main experiment, the maximum dose level for treatment was selected in agreement with the Study Protocol.

Dose levels of 5000, 2500, 1250, 625, 313, 156, 78.1 39.1 µg/mL were used both in the absence and presence of S9 metabolism.

Based on the cytotoxicity observed in the first experiment, dose levels of 4000, 3300, 2780, 2310, 1930, 1610, 1340, 1120, 930 and 775 µg/mL were selected for the second main experiment.

Test item solutions were prepared in sterile water for injection.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin-B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels were selected for the scoring of micro-nucleated cells on the basis of the cytotoxicity of the test item treatments calculated by thecytokinesis-block proliferation index (CBPI).

               S9           Treatment time (hours)      Harvest time (hours)        Dose level (µg/mL)

                                                                                                                                                                              

1             -/+                    3                                           27              5000, 2500 and 1250

2             -                     26                                           26              4000, 3300 and 2780

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent negative control value was observed at any dose level in the first main experiment.

In the second main experiment, statistically significant increases in the incidence of micronucleated cells over the concurrent negative control value were observed at the high and intermediate dose levels selected for scoring. In addition, a dose-effect relationship was indicated and at the highest dose level, incidences exceeded the range of RTC historical values for negative controls in both replicate cultures.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide, Mitomycin-C and Colchicine, indicating the correct functioning of the test system.