(1-hydroxyethylidene)bisphosphonic acid, potassium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 12 2010 to June 28 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

genetic toxicity in vitro, other

Remarks:

Bacterial mutagenicity, mammalian mutagenicity and mammalian cytogenicity

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

See read-across justification in IUCLID Section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

No evidence of cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

No evidence of cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

No evidence of cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

No evidence of cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3450 µg/ml (free acid), 24 h exposure

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium Salts of (1-Hydroxyethylidene)bisphosphonic acid (2-3 Na:1)

EC Number:

701-238-4

Molecular formula:

HEDP-2Na C2H6Na2O7P2 HEDP-3Na C2H5Na3O7P2

IUPAC Name:

Sodium Salts of (1-Hydroxyethylidene)bisphosphonic acid (2-3 Na:1)

Test material form:

solid - liquid: aqueous solution

Method

Target gene:

Thymidine Kinase Locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphthoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I, with and without S9 mix: 575, 1150, 2300, 4600, 9200, 18400 µg/ml (equivalent to 556-1784.8 µg/ml active acid);
Experiment II, without S9 mix: 287.5, 575, 1150, 2300, 3450, 4600 µg/ml (equivalent to 27.9-446 µg/ml active acid)
with S9 mix: 656.3, 1312.5, 2625, 5250, 10500, 21000 µg/ml (equivalent to 64-2067 µg/ml active acid).
without S9 mix: 21000 µg/mL (equivalent to 2067 µg/ml active acid).

Following the expression phase of 48 hours, the cultures at the lowest concentrations of 575 µg/ml in experiment I with and without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. In experiment II the concentrations at 287.5 µg/mL without metabolic activation and 656.3 µg/mL with metabolic activation were not continued for the same reason.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility properties

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

ACTIVATION: Phenobarbital/beta-naphthoflavone induced rat liver S9, NADP and glucose-6-phosphate as cofactors, final concentration in medium:5 % (v/v)

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 4 hours and 24 hours without metabolic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/ml TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period:
- Exposure duration: first experiment: 4 hours treatment with and without metabolic activation; second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10-15 days

SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: duplicate cultures; negative result in first experiment confirmed in second experiment

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

OTHER EXAMINATIONS:
- Other: size distribution of colonies

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and/or vehicle controls and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth and the cloning efficiency are less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3450.0 µg/ml (free acid), 24 h exposure

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not affected
- Effects of osmolality: Not increased
- Evaporation from medium: Not examined
- Water solubility: 12 %
- Precipitation: No precipitation was observed by the unaided eye up to the maximum concentration.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The highest concentration level of a pure substance should be 10 mM (corresponding to 2.28 mg/ml of the active ingredient) unless limited by the toxicity of the test item (reduced cell culture growth and/or cloning efficiency). Thus the maximum concentration in the pre-experiment was 2.28 mg/ml of the active ingredient or 18.4 mg/mL of the test item as supplied based on a purity of 12.4%. Test item concentrations between 143.8 and 18400 µg/ml were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hours of treatment with and without metabolic activation. After 24 hours treatment strong toxic effects occurred at 4600 µg/mL and above.


COMPARISON WITH HISTORICAL CONTROL DATA: Performed


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 hours of treatment. In experiment II following 24 hours treatment without metabolic activation, cytotoxic effects as described above occurred at 3450 and 4600 µg/mL. Based on the steep cytotoxic gradient of the test item the relative total growth (RTG) of both parallel cultures fell short of the lower limit of approximately 10%. However, according to the current IWGT recommendations data at RTG levels between 1 and 10% can be used to support a non mutagenic result provided that the dose spacing was 2.0 or lower and no relevant increase of the mutation frequency is observed at RTG levels below 10%.

Any other information on results incl. tables

Summary Table

conditions	conc. µg/ml (aqueous salt solution	conc. µg/ml (free acid)	S9 mix	relative total growth	mutant colonies/ 10^6cells	threshold	relative total growth	mutant colonies/ 10^6cells	threshold
Experiment I / 4 h treatment				culture I			culture II
Solv. control with water			-	100.0	113	239	100.0	95	221
Pos. control with MMS	19.5		-	30.6	309	239	33.0	299	221
Test item	575.0	72.1	-	culture was not continued^#			culture was not continued^#
Test item	1150.0	144.1	-	71.7	254	239	156.4	115	221
Test item	2300.0	288.3	-	70.2	168	239	110.0	177	221
Test item	4600.0	576.6	-	61.0	226	239	136.8	149	221
Test item	9200.0	1153.2	-	86.3	165	239	98.2	131	221
Test item	18400.0	2306.3	-	74.3	184	239	85.6	170	221
Experiment I / 4 h treatment				culture I			culture II
Solv. control with water			+	100.0	165	291	100.0	139	265
Pos. control with CPA	3.0		+	79.1	340	291	49.5	144	265
Pos. control with CPA	4.5		+	30.7	311	291	23.4	344	265
Test item	575.0	72.1	+	culture was not continued^#			culture was not continued^#
Test item	1150.0	144.1	+	143.5	174	291	83.9	183	265
Test item	2300.0	288.3	+	102.5	179	291	88.1	92	265
Test item	4600.0	576.6	+	120.4	131	291	80.7	107	265
Test item	9200.0	1153.2	+	91.5	129	291	83.7	162	265
Test item	18400.0	2306.3	+	76.7	149	291	61.7	128	265
Experiment II / 24 h treatment				culture I			culture II
Solv. control with water			-	100.0	189	315	100.0	124	250
Pos. control with MMS	13.0		-	17.9	630	315	12.4	489	250
Test item	287.5	36.0	-	culture was not continued^#			culture was not continued^#
Test item	575.0	72.1	-	60.7	178	315	86.8	165	250
Test item	1150.0	144.1	-	66.5	149	315	94.9	170	250
Test item	2300.0	288.3	-	70.8	144	315	82.9	155	250
Test item		432.4	-	37.0	134	315	32.1	119	250
Test item	4600.0	576.6	-	5.6	102	315	2.8	211	250
Experiment II / 4 h treatment				culture I			culture II
Solv. control with water			+	100.0	173	299	100.0	141	267
Pos. control with CPA	3.0		+	45.7	332	299	35.8	497	267
Pos. control with CPA	4.5		+	51.7	357	299	50.1	427	267
Test item	656.3	82.3	+	culture was not continued^#			culture was not continued^#
Test item	1312.5	164.5	+	91.4	178	299	98.8	154	267
Test item	2625.0	329.0	+	82.9	194	299	74.4	163	267
Test item	5250.0	658.1	+	89.4	191	299	73.1	178	267
Test item	10500.0	1316.1	+	93.9	169	299	79.0	183	267
Test item	21000.0	2632.0	+	96.3	183	299	71.9	204	267
Experiment II / 4 h treatment				culture I			culture II
Solv. control with water			-	100.0	159	285	100.0	169	295
Pos. control with MMS	19.5		-	16.1	601	285	17.8	639	295
Test item	21000.0	2632.0	-	74.0	203	285	51.6	298	295

threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

#  culture was not continued since a minimum of four concentrations is required by the guidelines

 

Applicant's summary and conclusion

Conclusions:

In a mammalian cell mutagenicity assay (reliability score 1), conducted according to OECD Test Guideline 476 and in compliance with GLP, HEDP (2-3Na) (neutral pH) was assessed for its induction of mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the presence and absence of metabolic activation. No reproducible, dose-dependent increase in the number of mutations was observed in either the initial or repeat experiment. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.