Tetrasodium (1-hydroxyethylidene)bisphosphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For HEDP sodium salts, information is avaiable from in vitro (two Ames tests, one mouse lymphoma assay, one micronucleus test) and in vivo tests (micronucleus test, dominant-lethal-assay) is available. None of the studies gave any indication of genotoxic potential. A combined chronic toxicity / carcinogenicity study performed with the disodium salt of HEDP gave no rise to concern regarding genotoxic / carcinogenic effects.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 487 (in vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

EXPERIMENT I
- without S9 mix: 13.7, 24.1, 42.1, 73.7, 128.9, 225.7, 394.9, 691.1, 1209.4, 2116.4 µg/mL
- with S9 mix: 13.7, 24.1, 42.1, 73.7, 128.9, 225.7, 394.9, 691.1, 1209.4, 2116.4 µg/mL

EXPERIMENT II
- without S9 mix: 13.7, 24.1, 42.1, 73.7, 128.9, 225.7, 394.9, 691.1, 1209.4, 2116.4 µg/mL

Vehicle / solvent:

Deionised water

Untreated negative controls:

other: solvent control served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 - pulse treatment

Untreated negative controls:

other: solvent control served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: demecolcin

Remarks:

without S9 - continuous treatment

Untreated negative controls:

other: solvent control served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

HUMAN LYMPHOCYTES
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (29 years old) for Experiment I and from a male donor (31 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

CULTURE CONDITIONS
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Evaluation criteria:

Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culturewere scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI is determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

CBPI=(MONCx1)+(BINCx2)+(MUNCx3)/n

CBPI Cytokinesis-block proliferation index
n Total number of cells
MONC Mononucleate cells
BINC Binucleate cells
MUNC Multinucleate cells

Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]

T Test item
C Solvent control

Statistics:

Statistical significance will be confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control.

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

In both experiments no precipitation of the test item in the culture medium was observed.

No relevant influence on osmolarity was observed. The pH was adjusted to physiological values.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

No relevant increase in micronucleated cells was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, at a concentration of 691.1 μg/mL a statistically significant increase in the number of micronucleated cells (0.75 %) was observed after treatment with the test item. The value lies within the range of the laboratory historical control data (0.15 – 1.70 % micronucleated cells) and can be estimated as biologically irrelevant.

In both experiments, either Demecolcin (100.0 ng/mL), MMC (2.0 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Experiment	Exposure	Conc. [µg/mL]	CBPI	Cytostasis [%]	Micronucleated cells [%]
I	4 h -S9	SC	1.88		0.30
I	4 h -S9	PC	1.14	84.7	10.30
I	4 h -S9	691.1	1.67	24.4	0.10
I	4 h -S9	1209.4	1.79	10.0	0.50
I	4 h -S9	2116.4	1.75	14.7	0.35
II	20 h -S9	SC	1.51		1.35
II	20 h -S9	PC	1.44	12.5	5.45
II	20 h -S9	394.9	1.79	nc	0.55
II	20 h -S9	691.1	1.60	nc	0.60
II	20 h -S9	1209.4	1.53	nc	0.20
I	4 h +S9	SC	1.92		0.30
I	4 h +S9	PC	1.46	50.3	4.85
I	4 h +S9	691.1	1.95	nc	0.75
I	4 h +S9	1209.4	1.91	1.2	0.70
I	4 h +S9	2116.4	1.91	0.7	0.30

SC = Solvent control

PC = Positive control

CBPI = Proliferation index

nc = not calculated (CBPI equal or higher than solvent control value)

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, SAT 140001 is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required or evaluable concentrations.

Executive summary:

The test item SAT 140001, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9		With S9
	Exp. I	Exp. II	Exp. I
Stimulation period	48 h	48 h	48 h
Exposure period	4 h	20 h	4 h
Recovery	16 h	-	16 h
Cytochalasin B exposure	20 h	20 h	20 h
Total culture period	88 h	88 h	88 h

In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2116.4 μg/mL of the test item) was chosen with regard to the purity (94.5%) of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

No relevant increase in micronucleated cells was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, at a concentration of 691.1 μg/mL a statistically significant increase in the number of micronucleated cells (0.75 %) was observed after treatment with the test item. The value lies within the range of the laboratory historical control data (0.15 – 1.70 % micronucleated cells) and can be estimated as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, SAT 140001 is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required or evaluable concentrations.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

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

Qualifier:

according to guideline

Guideline:

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

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

Target gene:

Thymidine Kinase Locus

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

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

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)

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 applicable

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%.

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

 

Conclusions:

HEDP x-Na (neutral pH) has been tested in a mammalian cell mutagenicity assay conducted according to OECD 476 and under GLP. No reproducible, dose-dependent increase in the number of mutations was observed in either the initial or repeat experiment In conclusion it can be stated that the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the presence and absence of metabolic activation.It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.

Executive summary:

The study was performed to investigate the potential of HEDP x-sodium salt (neutral pH) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. An additional experimental part was added to the second experiment with 4h treatment without metabolic activation to adopt the experimental part of 4 hour treatment without metabolic activation to the modified molecular weight of 228 g/mol. Only one concentration (21000 µg/ml equal to about 10 mM) was tested in the additional experiment.

The maximum concentration level of the test item should be 10 mM (corresponding to 18.4 (based on a molecular weight of 206 g/mol) or 21.0 mg/ml (based on a molecular weight of 263.2 g/mol) of the active ingredient) unless limited by the cytotoxicity of the test item. Thus the maximum concentration in the pre-experiment and in the first experiment was 18.4 mg/ml of the test item as supplied based on a purity of 12.4% and a molecular weight of 228 g/mol. On request of the sponsor the molecular weight of the test item was corrected to263.2 g/mol to reflect the true conditions at neutral pH-values. The maximum concentration in the second experiment was consequently increased to 21.0 mg/ml in the experimental parts using 4 hours treatment with and without metabolic activation. Following 24 hours treatment without metabolic activation the concentration range was limited by cytotoxic effects.

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%.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments up to the maximum concentration with and without metabolic activation. The threshold of 126 plus each solvent control count and the historical range of solvent controls were exceeded in culture I of the first experiment at the lowest concentration of 1150 µg/mL without metabolic activation and in the second culture of the second experiment at 21000 µg/mL, 4 hours treatment without metabolic activation. However, no comparable increase was noted in the parallel cultures under identical conditions. Therefore, the isolated increases described above are judged as irrelevant fluctuations. In experiment I the range of the historical solvent control data was exceeded at 4600 µg/mL at culture I without metabolic activation. Again, this effect was not reproduced in the parallel culture performed under identical experimental conditions and therefore, considered irrelevant.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT^â11statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in the first culture of the second experiment without metabolic activation and in the second culture of the second experiment with metabolic activation. The trend noted in the second experiment without metabolic activation was judged as irrelevant since it actually was reciprocal, going down versus increasing concentrations. Even the trend noted in the second experiment with metabolic activation was judged as irrelevant since the actual levels of the mutation frequency remained within the historical range of solvent controls and the threshold was not exceeded.

In this study the range of the solvent controls was from 95 up to 189 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 92 up to 298 mutant colonies per 10⁶cells. The highest solvent control values (189 and 173 colonies per 10⁶cells) exceeded the recommended 50 – 170 x 10⁶control range as stated under paragraph 8.12, acceptability of the assay, of this report. The mean values of both parallel cultures however, (157 and 156.5 colonies per 10⁶cells) are fully acceptable. The cloning efficiency exceeded the upper limit of the acceptance criteria somewhat in the second culture of the second experiment without metabolic activation. The data are acceptable however, since the cloning efficiency of the parallel culture remained within the acceptable range.

MMS (19.5 µg/ml following 4 hours treatment and 13.0 µg/mL following 24 hours treatment) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Deionised water

Untreated negative controls:

yes

Remarks:

concurrent untreated control

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (without S9 Mix); 2-aminoanthracene (with S9 Mix)

Details on test system and experimental conditions:

Salmonella typhimurium; strain TA 102; genotype his G 428, rfa-, uvrB+, R-factor; type of mutations: base-pair substitutions

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

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

RESULTS

The test item SAT 140001 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella lyphimurium strain TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate.

The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers was observed following treatment with SAT 140001 in strain TA 102 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

SUMMARY EXPERIMENT I

metabolic activation	test group	dose per plate [µg]	mean colony counts	SD
without	water (solvent control)	-	486	+/- 22
	untreated control	-	470	+/- 14
	test material	3	467	+/- 25
	test material	10	475	+/- 34
	test material	33	481	+/- 10
	test material	100	502	+/- 16
	test material	333	483	+/- 17
	test material	1000	491	+/- 15
	test material	2500	490	+/- 11
	test material	5000	525	+/- 7
	MMS (positive control)	2.0 µL	5235	+/- 450
with	water (solvent control)	-	625	+/- 12
	untreated control	-	625	+/- 10
	test material	3	615	+/- 15
	test material	10	631	+/- 14
	test material	33	640	+/- 32
	test material	100	663	+/- 13
	test material	333	587	+/- 32
	test material	1000	582	+/- 28
	test material	2500	571	+/- 12
	test material	5000	574	+/- 14
	2 -AA (positive control)	10.0 µg	1284	+/- 8

SUMMARY EXPERIMENT II

metabolic activation	test group	dose per plate [µg]	mean colony counts	SD
without	water (solvent control)	-	480	+/- 6
	untreated control	-	453	+/- 22
	test material	3	454	+/- 12
	test material	10	475	+/- 28
	test material	33	486	+/- 16
	test material	100	510	+/- 4
	test material	333	473	+/- 9
	test material	1000	492	+/- 12
	test material	2500	508	+/- 10
	test material	5000	501	+/- 24
	MMS (positive control)	2.0 µL	4983	+/- 269
with	water (solvent control)	-	620	+/- 8
	untreated control	-	642	+/- 39
	test material	3	643	+/- 21
	test material	10	664	+/- 27
	test material	33	660	+/- 30
	test material	100	697	+/- 36
	test material	333	628	+/- 26
	test material	1000	629	+/- 16
	test material	2500	616	+/- 42
	test material	5000	590	+/- 12
	2 -AA (positive control)	10.0 µg	1306	+/- 11

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strain used. Therefore, the tested material is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
The result has to be considered in combination with the earlier submitted bacterial reverse mutation assays and was conducted to provide information on the fith missing strain (TA 102).

Executive summary:

This study was performed to investigate the potential of SAT140001 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strain TA 102. The assay was performed with and without liver microsomal activation.

Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiments:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers was observed following treatment with SAT140001 in strain TA 102 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: Ames Test

Principles of method if other than guideline:

The test was performed according to Ames et al. (Mutation Research 31 (1975) 347-364), Methods for detecting carcinogens and mutagens wlth the salmonella/mammalian microsome mutagenicity test. Guideline was not available at the time the test was performed.

GLP compliance:

no

Remarks:

pre GLP

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced and Phenbarbitol-induced rat liver S9

Test concentrations with justification for top dose:

Test substance: 2.7 / 27 / 270 / 2700 µg/plate
Positive controls: 67.5 µg/plate (o-nitro-p-phenylendiamin, 270 µg/plate (p-toluol-sulfonic acid hydrazide)
Solvent controls: 100 µl H2O + 100 µl acetone or 100 µl H2O + 100 µl DMSO, resp.
Untreated controls

Vehicle / solvent:

Water/acetone or water/DMSO, resp.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water/acetone or water/DMSO, resp.

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: o-Nitro-p-phenylendiamine

Remarks:

Strains TA 1537, TA 1538, TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water/acetone or water/DMSO, resp.

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: p-toluolsulfonamidehydrazide

Remarks:

TA 100 and TA 1535

Evaluation criteria:

A substance is considered positive if at least one of the tested concentrations results in 2.5-times increase in the number of visible colonies relative to the solvent control in at least one test strains.

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, 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

Additional information on results:

Activity of Aroclor induced S9 was confirmed by testing TA 98 with Benzo-a-pyrene: 580 back mutations were induced. TA 100 induced 720 back mutations with 2-anthramine

Conclusions:

HEDP-2Na has been tested for reverse mutation in bacteria, using a method that is similar to OECD 471. No evidence for mutagenicity was obtained under the conditions of the study up to 2700 µg/plate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

1974-02-04 to 1974-09-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted using methods comparable to OECD guideline 478 "Genetic Toxicology: Rodent Dominant Lethal Test". Not GLP. Results of mating are reported for each male, but not in detail for each female. A positive control was not included, but is not necessarily required according to the OECD guideline.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

yes

Remarks:

Not GLP. Results of mating are reported for each male, but not in detail for each female. A positive control was not included, but is not necessarily required according to the OECD guideline.

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

other: C3D2F1/J

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, Maine
- Age at study initiation: sexually mature
- Weight at study initiation: N/A
- Assigned to test groups randomly: Yes, on the basis of body weight
- Fasting period before study: N/A
- Housing: N/A
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): N/A
- Humidity (%): N/A
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Water
- Amount of vehicle (if gavage or dermal): 0.25 ml

Details on exposure:

Each of three groups of mice received the test substance (as a single oral daily dose in 0.25 ml volumes), at levels of 1000, 200, and 20 mg/kg bw for five days. One other group received water for five days. The fifth group received no treatment.

Duration of treatment / exposure:

5 d

Frequency of treatment:

once daily

Post exposure period:

N/A

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

total implants, resorptions and dead embryos

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The top dose for the dominant lethal assay was based on the maximum tolerated dose determined from a preliminary study (see below under additional information on results). The intermediate dosing level was arbitrarily set at 200 mg/kg and the lower test group received the human-usage level of 20 mg/kg bw.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): N/A

DETAILS OF SLIDE PREPARATION: N/A

METHOD OF ANALYSIS: N/A

OTHER: N/A

Evaluation criteria:

N/A

Statistics:

Data from experimental groups were compared to data from control groups using the Chi-square test for significant deviations (P<= 0.05, DF=1)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 62.5-2000 mg/kg bw
- Solubility: N/A
- Clinical signs of toxicity in test animals: 2 out of 10 mice died at the 2000 mg/kg dose level.
- Evidence of cytotoxicity in tissue analyzed: N/A
- Rationale for exposure: To determine an LD50 and/or a maximum tolerated dose for mice.
- Harvest times: Animals were exposed daily for 5 consecutive days.
- High dose with and without activation: 2000 mg/kg without activation
- Other: An LD50 could not be calculated, but the maximum tolerated dose was chosen as 1000 mg/kg.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): N/A
- Induction of micronuclei (for Micronucleus assay): N/A
- Ratio of PCE/NCE (for Micronucleus assay): N/A
- Appropriateness of dose levels and route: Top dose was based on maximum tolerated dose.
- Statistical evaluation: N/A

Total litter losses occurred very rarely throughout the study and they showed no relationship to dosage of the test substance. Only two litters approached being totally lost in all test groups. One incident occurred in the untreated controls in which two to three implants in one female were dead and another occurred in the high dose group (1000 mg/kg) in which only one of five implants was alive at necropsy. Both litter losses originated during the third week of mating and were attributed to a malfunction in the individual animals and therefore were not considered test-related.

The number of deaths in females mated with males given the test substance did not differ significantly from those in concurrent controls for any of the mating periods.

Mating performance of all test groups compared favorably with the controls. As in the examination of individual female data, individual poor performance of the sire which was not test related could easily be detected. For example one male in the mid test group failed to impregnate a female until the fifth week of mating. In the final two weeks of the study, three of the four females mated with this male mouse conceived but only carried an average of six implants per pregnancy. The average number of implants for the rest of the group was 8.9 for the entire study and 9.0 for all other sires combined during the same two weeks of mating. The fact that eighteen implants sired by the male mouse mentioned above produced no fetal deaths provides additional evidence supporting the decision that the performance of this male was not test related.

When it came to conception rates, implants and fetal mortality, little variation occurred between test groups and controls for any of the parameters measured.

The mutagenic index was measured. It was a calculation based on two independent variables, fetal deaths and total implants, used as a convenient comparison of group to group performance. No significant differences (P= 0.05. D.F=1) were found when test group mutagenic indices were compared to either the vehicle or untreated controls.

Mating Performance and Uterine Content Evaluation by Groups 

 

Group	Percent Pregnant	Implants Total / Average	Fetal Deaths Total / Average	Percent Resorption
Untreated Control	83.3	1675 / 8.37	78 / 0.39	4.47
Vehicle Control	78.7	1635 / 8.65	64 / 0.37	3.66
EHDP 1.000 mg/kg	80.0	1618 / 8.42	74 / 0.39	4.14
EHDP 200 mg/kg	73.9	1545 / 8.78	66 / 0.38	4.27
EHDP 20 mg/kg	73.1	1543 / 8.82	53 / 0.30	3.30

 

EHDP Dominant Lethal Assay Mutagenic Indices

 

Week Treatment	Untreated Control	Vehicle Control	EHDP  1000 mg/kg	EHDP  200 mg/kg	EHDP  20 mg/kg
1	3.88	4.76	4.27	4.18	4.13
2	2.37	4.10	3.85	5.85	2.94
3	4.37	4.78	5.70	3.83	5.43
4	5.57	4.10	3.66	4.49	3.76
5	5.15	2.80	7.41	2.81	2.61
6	6.15	3.16	3.41	5.21	2.69

 

 

Resorptions + Dead Embryos   x 100 = Mutagenic Index

       Total Implantations

Conclusions:

The dominant lethal assay on EHDP proceeded very smoothly and gave no indication of any potential genetic hazard at test levels up to maximum tolerated doses. The test substance was tested in the dominant lethal assay using male mice that were orally dosed from 20 mg to 1000 mg/kg once daily for five days. No test substance related variations relative to control values in conception rates, total implant averages, fetal death averages, resorption percentage and mutagenic indices were observed. Under the conditions of the study, the test substance was considered negative in the dominant lethal assay.

Executive summary:

Phosphonic acid, P,'-(1 -hydroxyethylidene)bis-,sodium salt was administered orally (0.25 ml volumes) to male mice of the C3D2F1/J strain at three dose levels (1000, 200 and 20 mg/kg) for five consecutive days. The study also included a vehicle control (water) and an untreated group. 20 mice were assigned to each group. Immediately following treatment, each male was caged with two untreated females for a period seven days and with two fresh females the following week. This procedure was continued for a total of six weeks, thereby encompassing the entire spermatogenic cycle of the mouse, which was purported to be 35 days. Consequently, each male was mated to twelve females over a six-week period: A total of 240 matings per dose group. On day 13 or 14 of gestation (as measured from the mid-week of presumptive mating), the females were sacrificed. Total implants, resorptions and dead embryos were enumerated and recorded. Various parameters such as average number of implants per female, average number of fetal deaths/female, fetal deaths per sire, and percent pregnancy per group were subjected to statistical analysis. The test substance produced data that were not significantly different from control values in conception rates, total implant averages, fetal death averages, resorption percentage and mutagenic indices. Therefore, the test substance was considered to be non-mutagenic when administered orally at maximum tolerated doses in the test system employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For the induction of genotoxicity, two principle mechanistic actions can be distinguished: the potential to induce either mainly gene- or chromosome mutations. For the conclusion on the mutagenic potential of a substance, both modes of action have to be investigated with suitable test systems.

 

Concerning the induction of gene mutations, two Ames tests (Klimisch 2) and one mouse lymphoma study (Klimisch 1) are available for sodium salts of HEDP. The Ames tests followed the principles of the OECD guideline 471, but were conducted in the 1970s, where GLP principles have not yet been established. Furthermore, they did neither include the Salmonella typhimurium tester strain TA 102 nor an equivalent Escherichia coli strain to evaluate the induction of DNA crosslinking. Therefore, another Ames test according to OECD guideline 471 and GLP principles was conducted with the strain TA 102 to address this specific question. No indication for a genotoxic potential was observed in this test either. The hypothesis that HEDP salts do not induce gene mutations is not only supported by the two already available Ames tests, but also by a fully OECD- and GLP-compliant mouse lymphoma assay conducted in 2010, that gave no indication for mutagenicity.

 

With regard to the induction of chromosome aberrations, information from two in vitro studies (mouse lymphoma assay, Klimisch 1; micronucleus test, Klimisch 1) and two in vivo tests (dominant-lethal-assay, Klimisch 2; micronucleus test, Klimisch 4) is available.

Though the main focus of the mouse lymphoma assay lies on the identification of gene mutations, it is also suited to detect chromosome aberrations. According to the OECD guideline 476, these could be differentiated from the gene mutations by their smaller colony size. As no dose-related, reproducible increase in the mutant frequency occurred at all in the mouse lymphoma test with the sodium salt of HEDP, it can be concluded that the substance did neither induce gene-, nor chromosome mutations under the conditions of the test.

The potential to induce micronuclei was investigated in an in vitro study with human lymphocytes following OECD guideline 487. In two independent experiments, the substance did not induce a relevant increase of micronucleated cells in the absence (exposure period: 4 and 20 h) and presence of S9 mix (exposure period: 4 h). The only observed significant induction of micronuclei occurred at the lowest concentration in experiment I with S9 mix, but the value was within the historical control and thus considered as biologically irrelevant. Neither precipitation nor cytotoxicity was detected up to the highest dose level.

The in vivo micronucleus test included several deviations from current OECD standards (2 instead of 3 doses; highest dose tested lower than MTD; 4 instead of 5 animals per dose; bone marrow samples collected after 6 h, not after 18-24 h). Though the sampling time was shorter than recommended in the guideline, the positive control induced a clear effect.

Another relevant in vivo study for the evaluation of chromosome mutations is the dominant-lethal-assay. The study reviews the chromosomal aberration potential in germ cells of male mice by observation of pre-and post-implantation losses. The formation of chromosomal alterations in germ cells is basically the same as that for somatic cells (e.g., deletions, inversions, translocations). As stated in the OECD Guideline 478, dominant lethals are generally accepted to be the result of chromosomal aberrations (structural and numerical anomalies), although intrauterine deaths may also be caused by gene mutations and reproduction toxic effects. Though the study was conducted prior to the adaption of the respective OECD and GLP guideline, it met the principles laid down in the current OECD guideline 478. However, a concurrent positive control was not included in the assay, but might according to the OECD guideline be omitted if positive control data are available from another dominant-lethal-assay in the same laboratory within the last 12 months. In the dominant-lethal assay, mice were dosed by gavage up to 1000 mg of HEDP-xNa/kg bw/d for 5 days. No statistically significant chromosomal aberrations were observed in any of the treatments. Under the conditions of the dominant-lethal-assay, HEDP-xNa did not induce cytogenic effects.

Taking into consideration the lack of effects seen in the two micronucleus assays and the in vitro mouse lymphoma test, it can be concluded that sodium salts of HEDP are not suspected to induce cytogenetic effects in vitro or in vivo. This is supported by negative results in the carcinogenicity study and the dominant lethal assay,

 

In conclusion, no potential to induce gene or chromosome mutations was observed in any of the in vitro- and in vivo genetic toxicity assays or the carcinogenicity study.

 

Justification for grouping:

See CSR Annex I or IUCLID section 13.

Justification for classification or non-classification

The available information on read across substances indicates that, when tested in vitro, HEDP 4-sodium salt does not cause mutagenicity in mammalian cells; this is supported by evidence for lack of bacterial mutagenicity from disodium HEDP. In addition, no evidence for cytogenicity was shown when disodium HEDP was tested in vitro or in vivo. Therefore it is concluded that classification for mutagenicity is not required for tetrasodium HEDP.