Reaction mass of [[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid and Phosphonic acid, P-[(tetrahydro-2-hydroxy-2-oxido-4H-1,4,2-oxazaphosphorin-4-yl)methyl]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

The study was performed between 10 July 2012 and 30 October 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The original study was reliability 1. Read-across to the registered substance is scientifically justified and is considered to be reliability 2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[[(2-hydroxyethyl)imino]bis(methylene)]-bisphosphonic acid, equilibrium mixture with 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

IUPAC Name:

[[(2-hydroxyethyl)imino]bis(methylene)]-bisphosphonic acid, equilibrium mixture with 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Constituent 2

Reference substance name:

[[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt

EC Number:

244-743-0

EC Name:

[[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt

Cas Number:

22036-78-8

Molecular formula:

C4H12NNaO7P2, C4H11NNa2O7P2 and C4H10NNa3O7P2 (Constituent 1, linear form) C4H12NNaO7P2 and C4H11NNa2O7P2 (Constituent 2, cyclic form) The molecular formula and molecular weights represented that of the speciated species of the sodium salt

IUPAC Name:

[[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Details on test material:

Sponsor's identification: [[(2-hydroxyethyl)imino]bis(methylene)]-bisphosphonic acid, equilibrium mixture with 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt
Description: White paste
Further information: the test substance contained approximately 11% water. The active acid equivalent is 71%. Analysis of the test substance indicates that it was composed of 57% cyclised : 43% linear forms.
CAS Number: 22036-78-8
Batch number: LE12579a
Date received: 03 April 2012
Expiry Date: 01 August 2013
Storage conditions: Room temperature, in the dark

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes

- Periodically checked for karyotype stability: no

- Periodically "cleansed" against high spontaneous background: yes

Cell Line :
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.

Cell Culture:
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.

Cell Cleansing:
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by
culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and
grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each
experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test
The dose levels of test item used were 10.63, 21.25, 42.5, 85, 170, 340, 680, 1360 and 2720 µg/ml, equivalent to 7.5, 15.1, 30.2, 60.4, 120.7, 241.4, 483, 966 and 1931 µg/ml active acid.

Mutagenicity Test - Experiment 1
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
4-hour without S9 0*, 85*, 170*, 340*, 680*, 1360*, 2720*, EMS 500* and 750*
4-hour with S9 (2%) 0*, 85*, 170*, 340*, 680*, 1360*, 2720*, DMBA 0.5* and 1*

Mutagenicity Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
24-hour without S9 0*, 85*, 170*, 340*, 680*, 1360*, 2720*, EMS 200* and 300*
4-hour with S9 (1%) 0*, 85*, 170*, 340*, 680*, 1360*, 2720*, DMBA 0.5* and 1*

* = Dose levels plated for mutant frequency
EMS = Ethyl methane sulphonate
DMBA = Dimethyl benzanthracene

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Hams F12 culture medium

The test item was accurately weighed and dissolved in Hams F12 culture medium and appropriate dilutions made. The molecular weight of the test item was calculated to be approximately 272 and therefore the maximum recommended dose was the 10mM concentration of 2720 µg/ml. The test item had a water content of 10.95% and an allowance was made for this when the test item formulations were prepared.

There was a change in pH of more than 1 pH unit when the test item was dosed into media but this was reduced to acceptable levels when Hepes was added to the media. Hepes is a common supplement added to culture media to aid its buffering capacity. The osmolality did not increase by more than 50 mOsm at the dose levels investigated.

The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethyl benzanthracene (DMBA)

Remarks:

EMA used in cultures without metabolic activation (S9). DMBA used in cultures with metabolic activation (S9).

Details on test system and experimental conditions:

PRELIMINARY CYTOTOXICITY TEST:
A preliminary cytotoxicity test was performed on cell cultures plated out at 1.5 x 10E6 cells/75 cm2 flask for the 4-hour exposure groups and at 1 x 10E6 cells/75 cm2 flask for the 24-hour exposure group, approximately 48 hours before dosing. On dosing, the growth media was removed and
replaced with serum free media (Hams F12) for the 4 hour exposure groups and Hams F12 with 1% FBS for the 24-hour exposure group. One flask
per dose level was treated with and without S9 metabolic activation, 9 dose levels using halving dilutions and vehicle controls were dosed. The dose
levels of test item used were 10.63, 21.25, 42.5, 85, 170, 340, 680, 1360 and 2720 µg/ml. Exposure was for 4 hours or 24 hours at 37°C,
after which the cultures were washed twice with phosphate buffered saline (PBS) before being trypsinised. Cells from each flask were suspended in
Hams F12 with 5% FBS, a sample was removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 ml of Hams F12 with 5% FBS and incubated for 6 to 7 days at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies.

Results from the preliminary cytotoxicity test were used to select the test item dose levels for the mutagenicity experiments.

MUTAGENICITY TEST:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient
cells for use in the test. For the 4-hour exposure groups of Experiment 1 cells were seeded at 1.5 x 10E6/75 cm2 flask approximately 48 hours
before being exposed to the test or control items. In Experiment 2 cells were seeded approximately 48 hours before being exposed to the test or
control items at 1.0 x 10E6/75 cm2 flask for the 24-hour exposure group and at 1.5 x 10E6/75 cm2 flask for the 4-hour exposure group in the
presence of S9. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with six dose levels of test item, and vehicle and positive controls. Treatment was for 4 hours in serum free media (Ham's F12) or for 24 hours in Hams F12 with 1% serum at 37°C in an
incubator with a humidified atmosphere of 5% CO2 in air. Hepes was added to the culture media for the exposure period only.

The dose range of test item was 85, 170, 340, 680, 1360 and 2720 µg/ml for the 4-hour exposure groups in the absence and presence of S9 and for the 24-hour exposure group of Experiment 2.

At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in Hams F12 with 5% FBS. A sample of
each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10E6 cells/flask in a 225 cm2 flask to allow
growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in
Hams F12 with 5% FBS and incubated at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air.

Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded
to estimate cytotoxicity.

During the 7 Day expression period the cultures were subcultured and maintained at 2 x 10E6 cells/225 cm2 flask on day 3 to maintain logarithmic
growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out
as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 ml of Hams F12 with 5% FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage
cloning efficiency for each dose group calculated.
ii) At 2 x 10E5 cells/75 cm2 flask (5 replicates per group) in Hams F12 with 5% FBS, supplemented with 10 µg/ml 6-Thioguanine (6-TG),
to determine mutant frequency. The flasks were incubated for 14 days at 37°C in an incubator with humidified atmosphere of 5% CO2 in air, then
fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.

The percentage of viability and mutation frequency per survivor were calculated for each dose group.

Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with
methanol and finally staining with a 10% Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and
without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with
its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance
criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the
scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10E-6. The
background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10E-6 will not be used for the evaluation of a test item.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test item clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly
elevated over the concurrent negative control.
iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced
the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice the solubility limit of the test article in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration
that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts.
To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed
duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

Evaluation criteria:

See assay acceptance criteria.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

modest reduction in cloning efficiency at 2720 µg/ml, equivalent to 1931 µg/ml active acid.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST:
A dose range of 10.63, 21.25, 42.5, 85, 170, 340, 680, 1360, 2720 µg/ml was used in the preliminary cytotoxicity test. The maximum
dose tested was the maximum recommended dose level, the 10mM concentration. An allowance for the water content (10.95%) of the test item was made when the test item formulations were prepared.

No precipitate of the test item was observed at the end of exposure in any of the exposure groups.

The results of the individual flask counts and their analysis are presented in Table 1 (attached background material). It can be seen that there was no dose-related reduction in the cloning efficiency (CE) in the 4-hour exposure groups in the presence or absence of S9. In the 24-hour exposure group there was a modest reduction in the cloning efficiency of 28% at the maximum dose tested when compared to the vehicle control

The maximum dose level for the main experiments was the maximum recommended dose level of 1270 µg/ml for all exposure groups in Experiment 1 and Experiment 2.
 
MUTAGENICITY TEST - EXPERIMENT 1:
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
4-hour without S9 0, 85, 170, 340, 680, 1360, 2720, EMS 500 and 750
4-hour with S9 (2%) 0, 85, 170, 340, 680, 1360, 2720, DMBA 0.5 and 1

No precipitate of the test item was seen at the end of exposure in either exposure group.

The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation exposure groups are presented in Table 2 and Table 3 (attached background material). There was no marked reduction in cloning efficiency demonstrated at Day 0 or Day 7 in either of the exposure groups.

The Day 0 and Day 7 vehicle control cloning efficiencies for the 4-hour exposure group in the absence of S9 did not achieve 70% in all the replicates, however since the value was at least 50% in all cases this was considered to be acceptable.

The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 2 and Table 3 (attached background material). There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 with or without the presence of S9.

It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.


MUTAGENICITY TEST - EXPERIMENT 2
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
24-hour without S9 0, 85, 170, 340, 680, 1360, 2720, EMS 200 and 3000
4-hour with S9 (1%) 0, 85, 170, 340, 680, 1360, 2720, DMBA 0.5 and 1

No precipitate of the test item was seen at the end of the exposure period in either exposure group.

The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation exposure groups are presented in Tables 4 and 5 (attached background material). It can be seen that, as in Experiment 1, there was no reduction in the Day 0 or Day 7 cloning efficiencies of the 4-hour exposure group in the presence of S9. The 24-hour exposure group demonstrated a modest reduction in cloning efficiency at 2720 µg/ml at the maximum dose tested when compared to the vehicle controls. This toxicity was similar to that seen in the Preliminary Toxicity Test for this dose group.

The Day 0 and day 7 vehicle control cloning efficiencies for both exposure groups did not achieve 70% cloning efficiency in all the replicates, however since they achieved at least 50% this was considered to be acceptable.

The mutation frequency counts and mean mutation frequency per survivor per 10E6 cells values are presented in Tables 4 and 5 (attached background material). There were no increases in mutation frequency per survivor that exceeded the vehicle control value by 20 x 10-6 in either exposure
group.

It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Remarks on result:

other: strain/cell type: (HPRT) locus of Chinese hamster ovary (CHO) cells.

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See attached background material for:

Table 1: Preliminary Cytotoxicity Results

Table 2: Experiment 1 - 4 Hour Exposure Without Metabolic Activation (S9)

Table 3: Experiment 1 - 4 Hour Exposure With Metabolic Activation (S9)

Table 4: Experiment 2 - 24 Hour Exposure Without Metabolic Activation (S9)

Table 5: Experiment 2 - 4 Hour Exposure With Metabolic Activation (S9)

Applicant's summary and conclusion

Conclusions:

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

HEBMP-xNa has been tested in a reliable study conducted according to OECD 476 and in compliance with GLP. The test substance did not induce any significant or dose-related increases in mutant frequency in either the presence or absence of metabolic activation in either of the two experiments. Appropriate positive and solvent controls were included and gave expected results. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Executive summary:

Introduction.

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476' In Vitro Mammalian Cell Gene Mutation Tests', Method B17 of Commission Regulation (EC) No 440/2008, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the US EPA OPPTS 870.5300 Guideline.. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Methods.

Chinese hamster ovary (CHO) cells were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4‑hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group	Final concentration of test item (µg/ml)
4-hour without S9	85, 170, 340, 680, 1360, 2720
4-hour with S9 (2%)	85, 170, 340, 680, 1360, 2720
24-hour without S9	85, 170, 340, 680, 1360, 2720
4-hour with S9 (1%)	85, 170, 340, 680, 1360, 2720

Results.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion.

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.