Calcium hydrogen phosphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-Sep-2012 to 06-Nov-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

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

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid

EC Number:

237-066-7

EC Name:

Phosphonic acid

Cas Number:

13598-36-2

Molecular formula:

H3O3P

IUPAC Name:

phosphonic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Phosphonic acid (Phosphorous acid)
- Substance type: White crystalline solid
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark desiccated

Method

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 µg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 10, 33, 100, 333, 820 µg/mL
Without S9-mix, 24 hours treatment: 10, 33, 100, 333, 820 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 0.3, 1, 3, 10, 33, 100, 333 and 820 µg/mL
With S9-mix, 3 hours treatment: 100, 300, 400, 500, 600, 700, 750 and 800 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 33, 100, 400, 500, 600, 700, 750 and 820 µg/mL
With S9-mix, 3 hours treatment: 33, 100, 300, 500, 600, 700, 750 and 820 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Exposure medium (RPMI 1640 Hepes buffered medium (Dutch modification) )
- Justification for choice of solvent/vehicle:

Test compound was soluble in exposure medium and exposure medium has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: cyclophosphamide 10 µg/mL

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is = 50 per 10^6 survivors and = 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH:
Solvent control: 7.4
820 µg/ml: 6.2
333 µg/ml: 6.9
- Effects of osmolality:
Solvent control: 0.284 mOsm/kg
820 µg/ml: 0.295 mOsm/kg
333 µg/ml: 0.291 mOsm/kg
- Precipitation: No precipitation was observed up to and including the top dose of 820 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed in the absence of S9, 3 hours treatment; Toxicity was observed at the dose level of 820 µg/mL in the presence of S9, 3 hours treatment and at dose level of 820 µg/mL in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Appropriate toxicity was observed up to and including the highest tested dose level in both experiments in the absence and presence of S9-mix.

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Phosphonic acid (Phosphorous acid) is considered not mutagenic in the absence and presence of S9-mix in the mouse lymphoma L5178Y test system

Executive summary:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Except the response of the solvent control cultures in the first experiment in the presence of S9-mix. Although these responses were below the lower limit of the range, clear negative results were obtained in this part of the study, the validity of the test was considered to be not affected.

 

Mutation frequencies in cultures treated with positive control chemicals were increased 8.6 and

10-fold for in the absence of S9-mix, and 14- and 17-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix after a 3 hours treatment period, Phosphonic acid (Phosphorous acid) did not induce a significant increase in the mutation frequency. However Phosphonic acid (Phosphorous acid) induced a 3.4-fold increase in the mutation frequency at the prolonged treatment period of 24 hours. The mutation frequency (213 x 10^-6mutant colonies) was above the global evaluation factor plus mutation frequency of the negative controls (GEF + MF_(controls): 190 x 10^-6) and outside the historical control data range. This increase was only observed at the highest tested dose level of 820 µg/ml with severe toxicity, a relative total growth of 7%.

 

In the presence of S9-mix, Phosphonic acid (Phosphorous acid) did not induce a significant increase in the mutation frequency in the first experiment. However Phosphonic acid (Phosphorous acid) induced a 5.1-fold increase in the mutation frequency in the repeat experiment. The mutation frequency (323 x 10^-6mutant colonies) was above the global evaluation factor plus mutation frequency of the negative controls (GEF + MF_(controls): 190 x 10^-6) and outside the historical control data range. This increase was only observed at the highest tested dose level of 820 µg/ml with severe toxicity, a relative total growth of 4%.

  

Since the RTG is below 10% both in the absence and presence of S9-mix, these concentrations are too toxic according to the guidelines and therefore the mutagenic responses are not biologically relevant. Furthermore, the next dose level of 750 µg/ml with a RTG of 17 and 32% in the absence and presence of S9-mix, respectively, did not even show a two-fold increase in the mutation frequency.

The dose- response was not seen as the dose levels of 700 and 750 µg/ml showed no increase in the mutation frequency more than the MF(_controls) + the GEF and were within the limit of the historical control data.Therefore, Phosphonic acid (Phosphorous acid) is considered not mutagenic in the absence and presence of S9-mix in the mouse lymphoma L5178Y test system.