Reaction mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt and 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from HEBMP-H, negative with and without activation in Salmonella typhimurium strains TA98 and TA100 (OECD Test Guideline 471) (SafePharm, 2003).
Cytogenicity in mammalian cells: negative with and without activation in peripheral human lymphocytes (OECD Test Guideline 473) (Harlan Laboratories, 2013b).
Mutagenicity in mammalian cells: negative with and without activation at HPRT locus in Chinese hamster ovary cells (OECD Test Guideline 476) (Harlan Laboratories, 2012f).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 22 June 2012 and 15 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Cell Culture:
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test 9Cell Growth Inhibition Test):
The dose range of test item used was 18.75, 37.51, 75.01, 150.02, 300.04, 600.09, 1200.18, 2400.35 and 4800.7 µg/ml, equivalent to 13.3, 26.6, 53.3, 106.5, 213.0, 426.1, 852.1, 1704.2 and 3408.5 µg/ml active acid.

Experiment 1
4(20)-hour without S9: 0*, 150, 300.1, 600.13*, 1200.25*, 2400.5* and 4801* µg/ml.
4(20)-hour with S9: 0*, 150, 300.1, 600.13*, 1200.25*, 2400.5* and 4801* µg/ml.

Experiment 2
24-hour without S9: 0*, 85, 170, 340, 680*, 1360* and 2720* µg/ml.
4(20)-hour with S9: 0*, 85, 170, 340, 680*, 1360* and 2720* µg/ml.


* Dose levels selected for metaphase analysis

Vehicle / solvent:

Vehicle: Eagle's minimal essential medium with HEPES buffer (MEM).

Justification for choice of solvent/vehicle:
The test item was accurately weighed, dissolved in Eagle's minimal essential medium with HEPES buffer (MEM) and serial dilutions prepared. A purity correction was applied prior to each experiment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9: (MMC)

Details on test system and experimental conditions:

METHODS OF APPLICATION:
In medium

DURATION
- Pre-incubation period:
48 hours
- Exposure duration:
Experiment 1 – 4 hours with and without S9. Experiment 2 – 24 hours without S9, 4 hours with S9.
- Expression time (cells in growth medium):
20 hours for 4 hours exposure
- Selection time (in incubation with a selective agent):
Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):
24 hours


SPINDLE INHIBITOR (Cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS:
Duplicate cultures

NUMBER OF CELLS EVALUATED:
100/culture

DETERMINATION OF CYTOTOXICITY
-Method:
Mitotic index – A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells

Evaluation criteria:

A positive response is recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-response relationship. For modest increases in aberration frequency, a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

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: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test item suspended in MEM
- Precipitation:

Preliminary toxicity test: No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, in any of the exposure groups.

Experiment 1: No precipitate of the test item was observed in the parallel blood cultures at the end of the exposure period, in any of the exposure groups. The molecular weight of the test item was originally calculated as 480.07; therefore the maximum dose level was 4801 µg/ml, which was calculated to be equivalent to 10 mM (maximum recommended dose level). However, this calculation was incorrect and, unfortunately, this error was translated into both the Preliminary Toxicity Test and Experiment 1. The slides from both the preliminary toxicity test and Experiment 1 had metaphases present at 4801 µg/ml (17.6 mM), thereby confirming that the test item at this concentration was not toxic. The next dose level down was 2400.5 µg/ml, which was equivalent to 9 mM and, therefore, very close to the 10 mM maximum dose level. As a consequence, the test item has been considerably over tested in these experiments. The molecular weight was re-calculated for Experiment 2 and was corrected to 272.08. Therefore, the maximum dose level in Experiment 2 was 2720 µg/ml (equivalent to the 10 mM maximum recommended dose level).

Experiment 2: No precipitate of the test item was observed in the parallel blood cultures at the end of the exposure period, in any of the exposure groups.The molecular weight was re-calculated for Experiment 2 and was corrected to 272.08. Therefore, the maximum dose level in Experiment 2 was 2720 µg/ml (equivalent to the 10 mM maximum recommended dose level).

- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test

ADDITIONAL INFORMATION ON CYTOTOXICITY: see Other information on results including tables

Preliminary Toxicity Test (Cell Growth Inhibition Test):

The molecular weight was erroneously calculated to be 480.07 at the start of the study. As a consequence, the test item was considerably over tested in this experiment with an upper dose level equivalent to 17.6 mM.

The mitotic index data are presented in Appendix 1 (5) and (6) (see attached background material - Appendix 1). It can be seen that the test item showed clear evidence of dose-related toxicity only in the 24-hour continuous exposure group at the upper end of the test item dose range and no toxicity was observed in the 4(20)-hour exposure groups. No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, in any of the exposure groups.

Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at the maximum dose level of 4800.7 µg/ml in all three of the exposure groups.

The dose levels for both Experiment 1 and 2 were based on the maximum recommended dose level in accordance with the OECD 473 test guideline.

Chromosome Aberration Test – Experiment 1

The dose levels (µg/ml) of the controls and the test item are given below:

4(20)-hour without S9: 0*, 150, 300.1, 600.13*, 1200.25*, 2400.5*, 4801*, MMC 0.4*

4(20)-hour with S9 (2%): 0*, 150, 300.1, 600.13*, 1200.25*, 2400.5*, 4801*, CP 5*

*: dose levels selected for metaphase analysis MMC:

Mitomycin C

CP: Cyclophosphamide

The molecular weight was erroneously calculated at the start of the study. As a consequence, the test item was considerably over tested in this experiment.

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum test item dose level of 4801 µg/ml in both the presence and absence of metabolic activation.

The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, Appendix 2 (see attached background material - Appendix 2). These data show growth inhibition of 54% was achieved at 600.13 µg/ml in the absence of S9, although there was moderate growth inhibition at 1200.25 and 2400.5 µg/ml (24% and 26%, respectively). However, there was no appreciable growth inhibition at 4801 µg/ml. As the 50% growth inhibition was exceeded at 600.13 µg/ml only and not exceeded at dose levels higher than this, the mitotic index value of 46% was considered acceptable because the test item showed no evidence of clastogenicity. There is no obvious reason why greater toxicity was observed at this intermediate dose level of 600.13 µg/ml. With no precipitate being recorded at any dose level it cannot be due to test item exposure variations. This and the fact no qualitative toxicity was observed in the preliminary toxicity test and a similar dose level in the 24-hour continuous exposure group exhibited no toxicity suggest the effect was spurious in nature. In the presence of S9, no growth inhibition was observed.

No precipitate of the test item was observed at the end of the treatment period.

The selection of the maximum dose level for metaphase analysis was based on the maximum dose level used in the experiment. The chromosome aberration data are given in Form 1, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9) even when tested up to 17.6 mM. The slides confirm the presence of scorable metaphases present at 4801 µg/ml, thereby confirming that the test item at this concentration is not toxic.

The polyploid cell frequency data are given in Form 1, Appendix 2 (see attached background material - Appendix 2). The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test – Experiment 2

The dose levels (µg/ml) of the controls and the test item are given below:

24-hour without S9: 0*, 85, 170, 340, 680*, 1360*, 2720*, MMC 0.2*

4(20)-hour with S9 (1%): 0*, 85, 170, 340, 680*, 1360*, 2720*, CP 5*

*: dose levels selected for metaphase analysis

MMC: Mitomycin C

CP: Cyclophosphamide In

In Experiment 2, the molecular weight was recalculated and was 272.08; therefore, the maximum dose level in Experiment 2 was 2720 µg/ml equivalent to the 10 mM maximum recommended dose level.

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 2720 µg/ml in both the absence and presence of S9.

The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 2, Appendix 2 (see attached background material - Appendix 2). These data show a growth inhibition of 43% was achieved at 2720 µg/ml in the absence of S9 and no growth inhibition in the presence of S9.

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

The selection of the maximum dose level for metaphase analysis was the same as Experiment 1 (based on the Maximum Recommended 10 mM Dose level), and was 2720 µg/ml in both the presence and absence of S9.

The chromosome aberration data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Conclusions:

HEBMP-xNa has been tested according to OECD Test Guideline 473 and in compliance with GLP. The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. Appropriate positive and solvent controls were included and gave expected results. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro under the conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

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

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.

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.

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.

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)

Conclusions:

HEBMP-xNa has been tested in a reliable study conducted according to OECD Test Guideline 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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2000

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

METI, MHLW and MAFF

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon (Salmonella strains); tryptophan operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

50-5000 µg active acid/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no information in study report

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation: 3 µg/plate TA 100, 5 µg/plate TA 1535, 2 µg/plate WP2uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation: 80 µg/plate TA 1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation: 2 µg/plate TA 98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 1 µg/plate TA 100, 2 µg/plate TA 1535 and TA 1537, 10 µg/plate E. coli WP2uvrA

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation: 5 µg/plate TA 98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): histidine/tryptophan deficient agar

NUMBER OF REPLICATIONS: Plated in triplicate, experiment repeated


DETERMINATION OF CYTOTOXICITY
- Method: other: condition of background lawn

METABOLIC ACTIVATION: S9 mix included glucose-6-phosphate, NADP and NADPH as co-factors. 0.5 ml of 10% S9 solution was added to 0.1 ml test material, 0.1 ml of bacterial culture and 2 ml of top agar - final concentration approx 2% S9.

Evaluation criteria:

A test material is considered positive if it produces a reproducible, dose-related and statistically significant increase in the revertant count of at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No test-specific confounding factors were reported, and no precipitation or toxicity were observed.

Table 1 Cytotoxicity assay, revertants per plate

+/- S9	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA 100	68	67	82	76	75	71	67	80	65	56	60
+		89	89	106	103	96	120	80	129	87	87	86*
-	WP” uvrA	20	24	22	15	20	16	20	22	17	28	23
+		24	21	30	37	30	30	21	13	41	28	39

* Sparse bacterial background lawn

Table 2 Experiment 1, plate incorporation, revertants per plate (mean of 3 plates)

Dose (µg/plate)	TA 100		TA1535		E. coli WP2 uvrA		TA 98		TA1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Negative control**	112	-	33	-	17	-	29	-	12	-
0*	99	102	33	16	29	23	20	33	13	17
50	93	99	33	16	21	24	14	30	11	16
150	96	94	41	17	23	25	21	23	14	14
500	114	97	28	15	22	25	20	34	12	15
1500	109	99	32	14	21	28	18	33	12	15
5000	95	75	31	16	24	26	19	24	12	15
Positive control	442	868	211	314	816	1002	166	27117	1177	466

* solvent control with water

** spontaneous reversion rate

Table 2 Experiment 2, plate incorporation, revertants per plate (mean of 3 plates)

Dose (µg/plate)	TA 100		TA1535		E. coli WP2 uvrA		TA 98		TA1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Negative control**	120	-	27	-	25	-	25	-	12	-
0*	108	123	31	14	27	34	24	39	12	14
50	122	124	29	11	19	28	22	35	13	17
150	127	124	32	21	27	28	22	38	17	15
500	117	120	36	17	31	35	25	39	12	19
1500	120	121	34	19	27	31	17	31	15	14
5000	113	109	33	22	28	34	22	45	16	17
Positive control	363	1232	251		615	1146	138	247	982	330

* solvent control with water

** spontaneous reversion rate

Conclusions:

HEBMP-H has been tested for mutagenicity to bacteria in a reliable assay conducted according to OECD Test Guideline 471 and in compliance with GLP. No increase in the number of revertants per plate was found with and without activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E.col WP2 uvrA in either the initial or the repeat plate incorporation assay. Appropriate positive, negative (spontaneous reversion) and solvent controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutation in bacteria under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data are available for bacterial mutagenicity for HEBMP-xNa. Therefore, bacterial mutagenicity data for the Category member HEBMP-H are read across. Data are available for HEBMP-xNa for in vitro cytogenicity and mutagenicity to mammalian cells.

HEBMP-H has been tested for mutagenicity to bacteria in a reliable assay conducted according to OECD Test Guideline 471 and in compliance with GLP. No increase in the number of revertants per plate was found with and without activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E.coli WP2 uvrA in either the initial or the repeat plate incorporation assay. Appropriate positive, negative (spontaneous reversion) and solvent controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutation in bacteria under the conditions of the test.

HEBMP-xNa has been tested according to OECD Test Guideline 473 and in compliance with GLP. The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. Appropriate positive and solvent controls were included and gave expected results. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro under the conditions of the test.

HEBMP-xNa has been tested in a reliable study conducted according to OECD Test Guideline 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.

In vivo testing is not required as the results of the in vitro studies were all negative.

Justification for classification or non-classification

Based on the available in vitro data for HEBMP-xNa and its parent acid, HEBMP-xNa does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.