[ethane-1,2-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): EDTMP-5Na: negative with and without activation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 (similar to OECD TG 471) (Gridley J., Ross W. D. 1981a).
Gene mutation (Bacterial reverse mutation assay / Ames test): EDTMP-xCayNa: negative with and without activation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 (similar to OECD TG 471) (Gridley J., Ross W. D. 1981b).
Gene mutation (Bacterial reverse mutation assay / Ames test): EDTMP-xNa: negative with and without activation in Salmonella typhimurium strain TA 102 (OECD TG 471) (Sokolowski, A (2012)).
Cytogenicity in mammalian cells: negative with and without activation in Chinese hamster ovary cells (similar to OECD TG 473) (Li A. P., Myers C. A. 1986).
Mutagenicity in mammalian cells: EDTMP-xNa: negative with and without activation in CHO cells (similar to OECD 473) (Li, A. P. 1986).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1977

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Only a summary report was available for review.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

range of strains tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

0.001, 0.01, 0.1, 1, 5 and 10 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: not stated in report

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA98 with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA100 and TA1538 with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Diethyl sulfate

Remarks:

TA1535 with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

None given

Statistics:

None stated in report

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no indication of toxic effects at the concentrations tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no indication of toxic effects at the concentrations tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

(Ethylenebis[nitrilobis(methylene)]]tetrakisphosphonic acid, potassium salt has been tested for mutagenicity to bacteria in a study with no recorded accordance to OECD 471 or being compliant to GLP. No evidence of a test related increase in the number of revertants was observed with or without activation. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The restrictions were that only four strains of bacteria were used.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains of bacteria used

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

1-1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given in report

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 and TA 100 without metabolic activation: 4 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA 98 and TA 100 with metabolic activation: 5 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: NaNO2 9 µg/plate

Remarks:

TA 1535 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without metabolic activation: 75 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminonthracene 5 µg/plate

Remarks:

TA 1535 and TA 1537 with metabolic activation

Details on test system and experimental conditions:

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

DURATION

- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: initial spot plate test; triplicate plates in repeat plate incorporation study

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of background lawn (plate incorporation); zones of inhibition of growth (spot test)

Evaluation criteria:

A positive response is indicated if three or more treatments are significantly greater than solvent control (p=0.01) and if there is a significant positive dose-response (p=0.001).

Statistics:

Treatment compared with solvent controls using a one-sided t-test and within-levels pooled variance

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Spot test: toxicity but no mutagenicity was observed in strains TA 98 and TA 100. No toxicity or mutagenicity was observed in strains TA 1535 and TA 1537.

Number of revertants per plate

Concentration (µg//plate)	TA 98			TA 100			TA 1535			TA 1537
	+ S9			+ S9			+ S9			+ S9
Solvent control	40	16	22	146	134	150	7	11	10	2	8	3
Negative control	41	-	-	170	-	-	13	-	-	9	-	-
10	22	36	39	95	93	96	8	6	4	5	12	6
3	22	14	33	99	70	100	6	6	4	7	3	6
1	38	35	23	128	134	102	5	5	6	6	7	4
0.2	37	40	30	145	149	139	5	5	10	7	6	2
0.04	42	55	36	142	152	165	8	5	6	7	6	5
0.01	31	29	57	128	135	142	11	7	8	5	6	6
Positive control	495	-	-	1078	-	-	178	-	-	166	-	-

 

 

Concentration (µg//plate)	TA 98			TA 100			TA 1535			TA 1537
	- S9			- S9			- S9			- S9
Solvent control	18	22	11	137	104	136	4	3	4	2	5	2
Negative control	22	-	-	150	-	-	11	-	-	6	-	-
1	12	12	5	150	121	142	4	3	4	2	5	2
0.3	13	20	17	141	123	123	7	12	5	3	4	7
0.1	22	26	13	135	136	104	6	4	8	5	4	2
0.2	20	16	15	181	153	141	9	6	7	8	5	4
0.004	27	15	19	150	136	139	4	5	12	8	5	2
0.001	20	17	23	94	107	97	3	10	10	4	5	4
Positive control	378	-	-	669	-	-	198	-	-	125	-	-

 

 

Conclusions:

EDTMP-xCaxNa has been tested according to a protocol that is similar to OECD 471. No increase in the number of revertants was observed when Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were tested up to cytotoxic concentrations with and without metabolic activation. Appropriate positive, negative and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29.07.1981 - 15.09.1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The deviation was that the number of strains used did not comply with the current guidelines.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains used

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Spot test 25 µl, plate incorporation 0.001 µl, 0.004 µl, 0.01 µl, 0.02 µl, 0.04 µl, 0.1 µl, 0.2 µl, 0.3 µl, 1 µl, 3 µl, 10 µl (stock concentration 500 µl/ml; volume per plate 20µl)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: High purity water or DMSO
- Justification for choice of solvent/vehicle: none given in report.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA98 and TA100 with metabolic activation 5 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene at 5 µg/ml and 7 µg/ml

Remarks:

TA1535 and TA1537 with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 and TA100 without metabolic activation 4 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium nitrite at 9 mg/ml

Remarks:

TA1535 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without metabolic activation 100 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates except for positive and negative controls where only one plate was tested, test not repeated


DETERMINATION OF CYTOTOXICITY
- Method: condition of background lawn

Evaluation criteria:

A positive response in the plate incorporation test is indicated if three or more treatments on the initial test (and/or retest, if performed) are significantly greater than solvent control (p=0.01) and if there is a significant positive dose response (p=0.01) for the initial test (and/or retest, if performed).

Statistics:

Bartlett's test

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Average number of revertants per plate

Concentration (µg/plate)	TA 98		TA 100		TA 1535		TA 1537
	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
Solvent control	32.0	40.0	145.6	153.6	13.3	13.6	6.3	12.3
Negative control	26.0	44.0	141.0	136	23.0	12.0	6.0	19.0
10	-	27.6	-	6.6	-	3.0	-	3.0
3	-	39.0	-	-	-	4.0	-	16.0
1	23.0	39.6	-	153.6	2.3	8.3	4.6	17.0
0.3	20.6	-	90.3		10.3	-	9.0	-
0.2	-	51.3	-	161.6	-	10.0	-	8.0
0.1	34.0	-	153.0		18.0	-	10.6	-
0.04	-	51.5	-	161.6	-	12.0	-	11.3
0.02	35.3	-	166.3		18.0	-	3.6	-
0.01	-	47,6	-	144.3	-	10.0	-	12.0
0.004	36.3	-	144.6		20.0	-	10.0	-
0.001	26.6	-	162.3		18.0	-	4.3	-
Positive control	639.0	569.0	1214.0	1310.0	570.0	307.0	622.0	256.0

Conclusions:

EDTMP-xNa has been tested in a study conducted according to a protocol that is similar to OECD 471. No evidence of mutagenicity to bacteria was found with or without microsomal activation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2012-07-18 to 2012-07-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Only one strain of bacteria was tested.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only one strain of bacteria tested

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

only one strain of bacteria tested

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation: methyl methane sulfonate, MMS used with Strain: TA 102 @ 3 µL/plate

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: 2-aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates. Initial plate incorporation assay was repeated using the pre-incubation method.


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

Phenobarbital/b-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of b-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 39.5 mg/ml (lot no. R 260412) in both experiments.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

number of revertants was reduced though background lawn condition was normal

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No precipitation of the test item occurred up to the highest investigated dose.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate in both independent experiments



Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the following concentrations (µg/plate):

Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 102 1000 - 5000 333 - 5000 2500, 5000 100 - 5000

Summary of Results Pre-Experiment and Experiment I Plate incorporation revertants per plate mean of three plates

Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
		Without Activation	With Activation
Solvent control (water)		355 ± 30	469 ± 21
Untreated		330 ± 6	497 ± 97
EDTMP, pH-neutral	3 µg	357 ± 26	498 ± 43
	10 µg	367 ± 6	446 ± 45
	33 µg	371 ± 17	430 ± 10
	100 µg	354 ± 22	263 ± 23
	333 µg	235 ± 24	35 ± 19
	1000 µg	10 ± 8	3 ± 3
	2500 µg	0 ± 1	0 ± 0
	5000 µg	0 ± 0	0 ± 0
Positive control	3.0 µl	2637 ± 381	1824 ± 268

Concentrations expressed as active acid content, Correction factor 1.25

Summary of Results Experiment 2 Pre-incubation revertants per plate mean of three plates

Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
		Without Activation	With Activation
Solvent control (water)		393 ± 28	554 ± 42
Untreated		384 ± 4	586 ± 9
EDTMP pH-neutral	3 µg	406 ± 22	626 ± 33
	10 µg	414 ± 7	613 ± 59
	33 µg	394 ± 19	552 ± 39
	100 µg	365 ± 26	231 ± 36
	333 µg	412 ± 12	33 ± 16
	1000 µg	369 ± 27	4 ± 2
	2500 µg	15 ± 2	2 ± 4
	5000 µg	1 ± 1	2 ± 3
Positive control	3.0 µl	3432 ± 58	1743 ± 284

Test substance concentrations expressed as active acid content, Correction factor 1.25

Conclusions:

EDTMP-xNa has been tested according to OECD 471 in Salmonella typhimurium strain TA 102 in compliance with GLP. No increase in the number of revertants was observed with or without activation in either the initial plate incorporation assay or the repeat experiment using pre-incubation. Appropriate positive, solvent and untreated controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Executive summary:

The test item EDTMP, pH-neutral 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 typhimurium 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:

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

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

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 102	1000 - 5000	333 - 5000	2500, 5000	100 - 5000

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with EDTMP, pH-neutral 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mammalian Bone Marrow Chromosome Aberration Test in rat (oral gavage study): EDTMP-H: negative (similar to OECD TG 475) (EG&G 2001).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May 08, 1981 to June 22, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline, with acceptable restrictions. The restrictions were that only 50 cells per animal were evaluated, and samples were collected only once. The study was conducted in compliance with GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

Only 50 cells/animal were evaluated instead of 100 cells. Animals were dosed for consecutive 5 days instead of single treatment and justification for same was not provided. Samples were collected only once instead of at least 2 times.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Outbred albino rats were obtained from Taconic farms
- Age at study initiation: Sexually mature
- Weight at study initiation: 125-200 g
- Assigned to test groups randomly: Yes, animals were randomized by weight
- Fasting period before study: Not reported
- Housing: Animals were housed individually.
- Diet: Food (Agway RMH 3000), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 7-9 d prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 69-75°F (mean)
- Humidity: 49-73% (mean)
- Air changes: Not reported
- Photoperiod: Not reported

IN-LIFE DATES: Not reported

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing solution of test substance was prepared in corn oil. The dosing solution was prepared freshly prior to treatment.

DOSE VOLUME: 1 ml/100 g bw

Duration of treatment / exposure:

5d

Frequency of treatment:

Once daily

Post exposure period:

22-24 h after last treatment

Remarks:

Doses / Concentrations:
0.24, 0.8 and 2.4 g/ kg bw/d
Basis:

No. of animals per sex per dose:

5 rats/sex/ group

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

- Positive control: Methylmethane sulfonate (MMS)
- Solvent used: Distilled water
- Route of administration: Oral gavage
- Doses / concentrations: 0.156 g/ kg bw/ d
- Dose volume: 1.5 ml/ 150 g bw
- Duration of dosing: Once daily for 5 d

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

SACRIFICE: Animals were sacrificed by cervical dislocation following CO2 anaesthesia after 2-4 h treatment with Colchicine.

TREATMENT AND SAMPLING: Animals were treated once daily for 5 consecutive days. Animals were treated with Colchicine (intraperitoneal; 1 mg/ kg) approx. 20 h after last treatment, to cause mitotic arrest of bone marrow cells. Bone marrow cells were collected 2-4 h after treatment with Colchicine. Bone marrow of both femurs was aspirated into 10cc syringe filled with prewarmed (37°C) 5 mL Hank’s balanced salt solution. After centrifugation (100x g), cells were suspended in 0.075 M KCl for 10 min at 37±1°C.

DETAILS OF SLIDE PREPARATION AND STAINING: 2-3 drops of cell suspension were placed onto microscopic slides and passed through flame. All slides were stained with Giemsa stain for 8 min and fixed with Carnoy’s fixative. Slides were washed in water (1 min) then with acetone (10-20 sec), then in acetone-xylene (1:1 v/ v; 10-20 sec) and finally in xylene for 5 min. Permount was used as the mounting and all slides were placed on slide warmer at 30-35°C for minimum 2 h before scoring.

METHOD OF ANALYSIS: 50 metaphase/ animal were analyzed. Cytogenetic abnormalities such as deletions, exchanges, rings, gaps and breaks were scored and mitotic index of each animal was determined. The vernier location for each cell scored was recorded.

Statistics:

Standard deviation (SD) and standard error (SE) was calculated.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

DETAILS ON CLINICAL SIGNS AND SYMPTOMS:

- Clinical signs: Animals treated with test substance revealed no signs of toxicity.

- Mortality: No mortality was observed in negative, vehicle and test substance treated groups.

RESULTS OF POSITIVE CONTROL:

- Clinical signs: Some of positive control group animals revealed signs of ataxia, inactivity, lethargy, weakness and hypersensitivity.

- Mortality: 4 animals of positive control group died during study.

Table 1. Bone marrow aberrations after treatment with Ethylenediamine tetraphosphoric acid and controls (Study # 25691)

Treatment	% of Total Cells Analyzed
	Total aberrations (including gaps)		Aberrations excluding gaps
	Male	Female	Male	Female
Distilled water	0.6	2.8	0	0.4
Corn oil	4.0	7.2	0	1.6
MMS(0.156 g/ kg bw)	18.0	38.5	9.0	15.4
Test substance (0.24 g/ kg bw)	3.6	4.4	0	0.4
Test substance (0.80 g/ kg bw)	2.0	4.8	0.4	0.8
Test substance (2.40 g/ kg bw)	4.0	2.8	0.4	0

MMS= Methylmethane sulfonate

Conclusions:

Ethylenediamine tetraphosphoric acid (CC base) has been tested in a chromosome aberration assay conducted according to a protocol that is similar to OECD 475 and under GLP conditions. The substance was administered orally at 0.24, 0.80 and 2.40 g/kg bw/d to albino Sprague Dawley rats for 5 days. No test substance treatment related mortalities were observed. Appropriate solvent (corn oil), negative (water) and positive controls were included and gave expected results. The test substance did not induce any chromosomal damage in the bone marrow cells of the treated rats. It is concluded that EDTMP-H is negative for the induction of chromosome aberrations in rat bone marrow under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Reliable data are available for EDTMP-xNa for mutagenicity to bacteria, and from in vitro mammalian gene cell mutation assay. Reliable data are available for EDTMP-H from an in vitro cytogenicity study; supporting data are available for EDTMP-H from in vitro bacterial and mammalian mutagenicity studies. Reliable data are available for EDTMP-xCaxNa for mutagenicity to bacteria. Supporting data are available for EDTMP-xK for mutagenicity to bacteria. Reliable data are available for EDTMP-H from an in vivo chromosome aberration study in rat (oral gavage administration).

In dilute aqueous conditions of defined pH, such as the ones in which in vitro studies are conducted, a salt will behave no differently to the parent acid, at identical concentration of the particular speciated form present, and will be fully dissociated to yield EDTMP-H and salt. Hence genetic toxicity properties for a salt (in contact with water or in aqueous media) can be directly read across to the parent acid and vice versa. In the present context the effect of the alkaline metal counter-ion (sodium) will not be significant and has been extensively discussed in the public literature. In biological systems and the environment, polyvalent metal ions will be present, and the phosphonate ions show very strong affinity to them.

EDTMP-5Na has been tested in a study conducted according to a protocol that is similar to OECD 471 (Monsanto 1981). No evidence of mutagenicity to bacteria was found with or without microsomal activation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538. Appropriate positive, negative and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test. The study is considered to be reliability 2.

EDTMP-xCaxNa has been tested according to a protocol that is similar to OECD 471. No increase in the number of revertants was observed when Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were tested up to cytotoxic concentrations with and without metabolic activation (Gridley J., Ross W. D. 1981). Appropriate positive, negative and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test. The study is considered to be reliability 2.

EDTMP-xNa has been tested according to OECD 471 in Salmonella typhimurium strain TA 102 in compliance with GLP (Sokolowski, A (2012)). No increase in the number of revertants was observed with or without activation in either the initial plate incorporation assay or the repeat experiment using pre-incubation. Appropriate positive, solvent and untreated controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Further supporting studies for mutagenicity to bacteria are available: EDTMP-H and EDTMP-xK have been tested and shown to be negative for mutagenicity to Salmonella strains TA98, TA100, TA1535, TA1537 and TA1538, with and without metabolic activation (Flowers 1976, Flowers 1977, both reliability 4).

EDTMP-H has been tested according to protocol that is similar to OECD 473 (Li A. P., Myers C. A. 1986). The study was considered to be reliability 2. No test substance induced increase in the incidence of chromosome aberrations in Chinese hamster ovary cells was observed with or without metabolic activation in either Lot A or Lot B. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosomal aberrations under the conditions of the test.

EDTMP-xNa has been tested in CHO cells for its potential to induce forward mutation according to a protocol that is similar to OECD 476 and in compliance with GLP (Li A. P. 1986). No evidence of mutagenicity was observed with or without metabolic activation in the initial or the repeat experiments. Appropriate positive and test medium controls were included and gave expected results. It is concluded that the test substance is negative for induction of forward mutations in CHO cells under the conditions of the test.

EDTMP-H has been tested in an in vivo chromosome aberration assay conducted according to a protocol that is similar to OECD 475 and under GLP conditions (EG&G 2001). The substance was administered orally at 0.24, 0.80 and 2.40 g/kg bw/d to albino Sprague Dawley rats for 5 days. No test substance treatment related mortalities were observed. Appropriate solvent (corn oil), negative (water) and positive controls were included and gave expected results. The test substance did not induce any chromosomal damage in the bone marrow cells of the treated rats. It is concluded that EDTMP-H is negative for the induction of chromosome aberrations in rat bone marrow under the conditions of the study.

Justification for classification or non-classification

Based on the available in vitro and in vivo genotoxicity data for the registration substance and its salts, EDTMP-H is not classified for mutagenicity according to Regulation (EC) No 1272/2008.