Sodium salts of [[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid (1-3 Na:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from DTPMP (5-7Na); negative with and without activation in Salmonella typhimurium TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (OECD Test Guideline 471 and in compliance with GLP) (Japan Oilstuff Inspectors Corporation, 2001a, reliability score 1).
Cytogenicity in mammalian cells: read-across from DTPMP (5-7Na); positive in Chinese hamster lung CHL/IU cells (OECD Test Guideline and in compliance with GLP 473) (Japan Oilstuff Inspectors Corporation, 2001b, reliability score 1).
Mutagenicity in mammalian cells: read-across from DTPMP (5-7Na); negative in mouse lymphoma L5278Y cells (similar to OECD Test Guideline 476 and in compliance with GLP) (Central Toxicology Laboratory, 1997, reliability score 2).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-Jun-2001 to 18-Sep-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemicals

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: CHL/IU (originally derived from female Chinese hamster lung)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimum essential medium
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
- Cells were purchased from Dainippon Pharmaceutical co.
- Cell suspension was mixed with ne tenth volume of DMSO and then frozen and stored in liquid nitrogen. The suspended cells were used after thawing and cultivation through up to 5 passages

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital- and 5,6-benzoflavone-induced male rat liver S9

Test concentrations with justification for top dose:

625-5000 µg mixed sodium salts/ml (pulse treatment)
150-3000 µg mixed sodium salts/ml (24-hour continuous treatment)
50-400 µg mixed sodium salts/ml (48-hour continuous treatment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle: solubility of test substance in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9, 0.1 µg/ml (pulse treatment), 0.03 µg/ml (continuous treatment)

Positive control substance:

mitomycin C

Remarks:

Mitomycin was dissolved in saline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9, 15 µg/ml (pulse treatment)

Positive control substance:

benzo(a)pyrene

Remarks:

Benzo(a)pyrene was dissolved Dimethyl Sulfoxide (DMSO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: pulse treatment 6 hours; continuous treatment, 24 or 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): pulse treatment 24 hours; continuous treatment 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.1 µg/ml, 2 hours
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 100 cells /culture, 2 cultures/treatment

DETERMINATION OF CYTOTOXICITY
- Method: other: cell density after crystal violet staining

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: mitotic index

Evaluation criteria:

Negative: both the incidence of aberrant cells (excluding gaps) and that of numerical aberrations <5%
Inconclusive: either of these incidences is 5-10%
Positive: either of these incidences >10%
A cell with at least one structural chromosomal aberration was classified as aberrant cell. The number of aberrant cells was counted in two different ways, one includes cells with no aberration other than gaps and another excludes these types of cells.

Statistics:

When the test was positive the D20 values were calculated from the test results. The D20 value was the concentration (mg/ml) required to induce any aberration in 20% of metaphases.

Key result

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

pulse treatment ( 6 and 24 hour exposure)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2500 µg mixed sodium salts/ml (pulse treatment),

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

48 hour treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=50 µg mixed sodium salts/ml (48-hour continuous treatment)

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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no precipitation observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- No, but cell survival was evaluated at 156-5000 µg mixed sodium salts/ml (pulse treatment, 24-hour continuous treatment and 48-hour continuous treatment) and the data used to determine the concentrations that were to be evaluated for chromosome aberrations

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cell survival:
- Pulse treatment -S9, µg mixed sodium salts/ml: 156 (109%), 313 (109%), 625 (97%), 1250 (96%), 2500 (76%), 5000 (58%)
- Pulse treatment +S9, µg mixed sodium salts/ml: 156 (98%), 313 (104%), 625 (103%), 1250 (108%), 2500 (102%), 5000 (77%)
- 24-hour continuous treatment -S9, µg mixed sodium salts/ml: 156 (115%), 313 (81%), 625 (47%), 1250 (37%), 2500 (19%), 5000 (0%)
- 48-hour continuous treatment -S9, µg mixed sodium salts/ml: 156 (50%), 313 (25%), 625 (7%), 1250 (5%), 2500 (1%), 5000 (0%)

Full details of the results from the 48 hours treatment (result reported as positive) are not presented in the study report. A graph of the results indicates that the incidence of cells with structural aberrations was 2.5% at 50 µg/ml; 5% at 100; 15% at 150; 37% at 200 and 49% at 300 µg/ml.

Table 1 Chromosome aberration test - treatment time 6 hours

Concentration µg/ml	+/- S9 mix	No. of cells analysed	Chromatid breaks	Chromatid exchanges	No. of cells with aberration (%)	No. of cells with gaps	Cell growth index (%)	Polyploid cells
Control	-	200	0	0	0	1	100	0
625	-	200	1	0	1	0	98	0
1250	-	200	3	1	4	0	91	0
2500	-	200	6	1	7	4	71	2
5000	-	200	9	1	9	3	41	0
Positive control	-	200	54	121	139	1	-	0
Control	+	200	0	0	1	0	100	0
625	+	200	0	2	2	1	91	0
1250	+	200	0	1	1	0	95	0
2500	+	200	3	3	4	0	99	0
5000	+	200	1	0	1	0	67	0
Positive control	+	200	7	42	46	1	-	0

 

Chromosome aberration test - treatment time 24 hours

Concentration µg/ml	+/- S9 mix	No. of cells analysed	Chromatid breaks	Chromatid exchanges	No. of cells with aberration (%)	No. of cells with gaps	Cell growth index (%)	Polyploid cells
Control	-	200	2	0	2	1	100	0
4.7	-	200	1	1	2	1	101	0
9.4	-	200	2	0	2	0	102	0
18.8	-	200	4	0	4	1	104	0
37.5	-	200	3	2	5	0	107	0
75	-	200	2	1	3	3	103	0
150	-	200	8	0	8	2	113	0
300	-	-	TOXIC
Positive control	-	200	30	30	58	0	-	0
Control	+	200	2	0	2	0	100	0
50	+	200	4	1	5	0	99	0
100	+	200	7	4	11	2	68	0
150	+	200	31	9	34	0	57	0
200	+	200	67	9	74	1	43	0
300	+	200	87	10	97	2	20	0
400	+	-	TOXIC
Positive control	+	200	47	56	86	0	-	0

 

Conclusions:

DTPMP (5-7Na) has been tested for clastogenicity in a valid in vitro mammalian chromosome aberration study in mammalian cell line CHL/IU with and without metabolic activation, conducted according to OECD Test Guideline 473 and in compliance with GLP. A dose-related increase in the number of cells with aberrations was observed after 48 hours treatment, without metabolic activation. No dose-depended increase in the number of cells with aberrations was observed after 6 or 24 hours treatment, with and without metabolic activation. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP (5-7Na) is positive for clastogenicity in vitro under the conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-Jul-2001 to 26-Jul-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

(only 2-AA used as positive control +S9)

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemicals

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Target gene:

histidine (Salmonella typhimurium)
tryptophan (Escherichia coli)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital- and 5,6-benzoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Test 1-1: 78-5000 µg mixed sodium salts/plate -S9, 156-5000 µg/plate +S9
Test 1-2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium
Test 2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium, 78-5000 µg mixed sodium salts/plate -S9 for E coli, 156-5000 µg mixed sodium salts/plate +S9 for all strains

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9; TA98, 0.1 µg/plate; TA100, 0.01 µg/plate

Positive control substance:

furylfuramide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 ; TA1535, 0.5 µg/plate

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9; TA1537, 80 µg/plate

Positive control substance:

9-aminoacridine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9, WP2 uvrA, 2 µg/plate

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9; TA98, 0.5 µg/plate; TA100, 1 µg/plate; TA1535 and TA1537, 2 µg/plate; WP2 uvrA, 10 µg/plate

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation method - the test substance, bacterial suspensions and buffer were mixed in a sterilized test tube and incubated with gentle shaking. After preincubation agar was added to this mixture and it was poured onto a plate. The plates were then incubated again.

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition

PRELIMINARY TEST
-A preliminary test was performed using a single plate per dose level.

Evaluation criteria:

Positive: mean number of revertant colonies more than double the negative control value and when it shows significant concentration-dependent increase. When the test substance is judged positive specific activity was calculated as the number of induced revertant colonies per 1 mg test substance.

Statistics:

No statistical analysis performed

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed from 1250 µg/plate with metabolic activation and from 625 without metabolic activation µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed from 1250 µg/plate with metabolic activation and from 313 µg/plate without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed from 1250 µg/plate with metabolic activation and from 625 µg/plate without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was observed from 625 µg/plate without metabolic activation, and wasn't observed with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed from 5000 µg/plate with metabolic activation and from 625 µg/plate without metabolic activation

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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no precipitation observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- Concentrations of 5-5000 µg/plate
- No growth inhibition in any strain
- Bacteriostatics observed at 1250 µg/plate -S9 in all strains
- No precipitation

COMPARISON WITH HISTORICAL CONTROL DATA:
- Negative/solvent control: within the range of historical data
- Positive controls: within the range of historical data

All treated cultures had less than 2-fold increase in mutant frequency and no evidence of a dose-response.  All positive controls gave a greater than 2-fold increase in mutant numbers.

Table 1 Experiment 1 Mean number of revertants (average of 3 plates)

 

Concentration (µg/plate)	+/- S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Control	-	102	10	23	14	7
78	-	102	8	21	16	9
156	-	88	7	18	16	6
313	-	71	5	18	8	1
625	-	0	0	7	0	0
1250	-	0	0	0	0	0
2500	-	0	0	0	0	0
5000	-	0	0	0	0	0
Positive control	-	528	422	631	559	194
Control	+	111	11	25	24	6
156	+	133	11	30	24	10
313	+	148	11	21	22	6
625	+	140	9	23	17	6
1250	+	122	6	21	11	3
2500	+	108	4	15	9	3
5000	+	98	5	9	8	2
Positive control	+	501	260	648	446	154
Mean number or revertants 16-19.07.2001

Table 2 Experiment 2 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)	+/- S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Control	-	104	11	-	16	7
20	-	101	6	-	16	8
39	-	94	8	-	12	8
78	-	106	8	-	14	8
156	-	97	9	-	14	5
313	-	81	6	-	14	4
1250	-	0	0	-	0	0
5000	-	0	0	-	0	0
Positive control	-	522	411	-	604	211

 

Table 3 Experiment 3 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)	+/- S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Control	-	99	8	20	21	7
20	-	105	10	-	18	7
39	-	107	11	-	19	6
78	-	110	10	22	19	6
156	-	103	11	14	17	7
313	-	86	8	16	15	4
625	-	-	-	13	-	-
1250	-	0	0	0	0	0
2500	-	-	-	0	-	-
5000	-	0	0	0	0	0
Positive control	-	510	419	655	601	183
Control	+	127	10	21	23	8
156	+	130	9	29	26	9
313	+	132	12	17	19	7
625	+	127	12	22	18	6
1250	+	114	8	15	14	7
2500	+	119	6	13	6	3
5000	+	103	6	12	3	2
Positive control	+	679	304	758	517	152

 

Conclusions:

DTPMP (5-7Na) has been tested for mutagenicity to bacteria in a valid gene mutation study in bacterial strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation, conducted according to OECD Test Guideline 473 and in compliance with GLP. No dose depended increase in the number of revertants was observed in any of the bacterial test strains when tested with or without metabolic activation up to cytotoxic concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP (5-7Na) is negative for mutagenicity in bacteria under the conditions of this test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17-Jan-1997 to 25-Feb-1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The test substance was not tested up to the maximum concentration required by guideline.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

yes

Remarks:

(not tested to maximum concentration required by guideline)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-1640
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital- and beta-naphthoflavone-induced male rat liver S9

Test concentrations with justification for top dose:

Phase 1: 266-2128 µg active acid/ml
Phase 2: 265-2121 µg active acid/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water for test substance, dimethyl sulphoxide used for positive control
- Justification for choice of solvent/vehicle: test substance supplied in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9; 750 µg active acid/ml

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9; 3 µg active acid/ml

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): no data

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: other: cell growth

Rationale for test conditions:

The L5178Y TK+/- mouse lymphoma mutation assay is designed to detect chemically induced gene mutation and/ or clastogenic effects in cells treated in culture by measuring forward mutation. The cells are an established cell line and are exposed to various concentrations of the test substance grown for the expression time and plated into microwells in the presence and absence of TFT to estimate the number of mutant cells per viable cell ( the mutant frequency). Since forward mutation can also be seen in solvent control cultures, the assay is based on the observation of an increased mutant frequency over and above that seen in the solvent control cultures. The cytotoxicity of the test substance is assessed by post treatment cloning efficiency.
Higher concentrations of the test substance produced excessive increases in the osmolality of the treatment medium. These increases in osmolality have been associated with artefactual responses in genotoxicity assays and therefore are considered inappropriate for testing.

Evaluation criteria:

- Criteria for a positive response: a statistically significant, dose related increase in mutant frequency is required, but not only at concentrations eliciting excessive toxicity. An associated absolute increase in mutant number above the solvent control values is a further requirement. Such a response must be reproducible in an independent experiment for the test substance to be described as positive in this assay.
- Criteria for a negative response: A negative response is obtained when there is no reproducible statistically significant dose related increase in mutant frequency. When reproducible significant increases in mutant frequency are seen only at levels of excessive toxicity, or when such increases are not accompanied by an increase in absolute numbers of mutants over solvent control values, then the effect is considered not to be indicative of a positive response in this assay.

Statistics:

- If considered necessary, the data are considered by logit regression, using a complimentary log-log link function. The dependent variable is the number of empty wells. This procedure provided maximum likelihood estimates of log mutant frequencies. Variances are inflated by the between duplicate heterogeneity factor.
- Tests for trend in log mutant frequency with actual concentration are obtained separately for each experiment, in the presence and absence of S9-mix. In addition, an overall test for trend combining data across experiments is performed.
- Intergroup comparisons of log mutant frequency comparing each treated group with the solvent control are performed within each experiment. In addition, tests for trend in log mutant frequency with actual concentration are obtained both within each experiment and combined across all experiments.
- All tests are one-sided. Similar analyses are carried out separately for the positive controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: test solutions neutralised to ~pH7
- Effects of osmolality: "higher concentrations [than 2200 µg mixed sodium salts/ml] produced excessive increases (up to 70 mM/kg) in the osmolality of the treatment medium. Such increases in osmolality have been associated with artefactual responses in genotoxicity assays ... and these concentrations were therefore considered not to be appropriate for testing."
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data

No increases in mutant frequency were seen in any of the tests and it was not considered necessary to conduct statistical analysis.  All the positive control cultures had elevated mutant frequencies as expected.

The maximum concentration tested was 2200 µg/ml.  Higher concentrations were claimed to give excessively high osmolarity, although the values given for 4256 and 4242µg/ml in subsequent tests only resulted in increases to 354 and 334 mOsm/kg respectively. Since no increases in mutant frequency were seen in the first test at a dose producing 330 mOsm/kg it could be argued that the dose of 4242 could have been tested.  All concentrations below 5000 µg/ml are <10 mM, the upper limit defined by OECD for this assay.  A toxicity limit was not reached in these tests - top levels had >75% survival.  Therefore the upper limit defined for this assay was not reached.

Conclusions:

DTPMP (5-7Na) has been tested for mutagenicity to mammalian cells in a valid gene mutation study in mouse lymphoma L5178Y cells with and without metabolic activation, conducted according to OECD Test Guideline 476 and in compliance with GLP. No increase in the mutant frequency was observed when tested with or without metabolic activation up to limit concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP (5-7Na) is negative for mutagenicity in mouse lymphoma L5178Y cells under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

DTPMP (5-7 Na) salt (EC:701-216-4) was tested in an in vivo mammalian alkaline comet assay, conducted according to OECD TG 489 and in compliance with GLP (Charles River Laboratories, 2022a, reliability 1). The substance was not genotoxic in liver, duodenum and stomach cells when sampled approximately 3-4 hours post dosing, in male rats that were dosed via oral gavage for two consecutive days up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines). No clinical signs of toxicity or mortality were observed in the treated animals.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are no available genetic toxicity data for DTPMP (1-3Na), therefore data for the Category members DTPMP (5-7Na) and DTPMP-H have been used.

See attachment to Section 13 for justification of read-across within DTPMP Category.

 

DTPMP (5-7Na) has been tested for mutagenicity to bacteria in a valid gene mutation study in bacterial strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation, conducted according to OECD Test Guideline 471 and in compliance with GLP. No dose-dependent increase in the number of revertants was observed in any of the bacterial test strains when tested with or without metabolic activation up to cytotoxic concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP (5-7Na) is negative for mutagenicity in bacteria under the conditions of this test (Japan Oilstuff Inspectors Corporation, 2001a, reliability score 1).

 

DTPMP (5-7Na) has been tested for clastogenicity in a valid in vitro mammalian chromosome aberration study in mammalian cell line CHL/IU with and without metabolic activation, conducted according to OECD Test Guideline 473 and in compliance with GLP. A dose-related increase in the number of cells with aberrations was observed after 48 hours treatment, without metabolic activation. A not dose-depended increase in the number of cells with aberrations was observed after 6 or 24 hours treatment, with and without metabolic activation. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP (5-7Na) is positive for clastogenicity in vitro under the conditions of this test (Japan Oilstuff Inspectors Corporation, 2001b, reliability score 1).

 

DTPMP (5-7Na) has been tested for mutagenicity to mammalian cells in a valid gene mutation study in mouse lymphoma L5178Y cells with and without metabolic activation, conducted according to OECD Test Guideline 476 and in compliance with GLP. No increase in the mutant frequency was observed when tested with or without metabolic activation up to limit concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP (5-7Na) is negative for mutagenicity in mouse lymphoma L5178Y cells under the conditions of this test (Central Toxicology Laboratory, 1997, reliability score 2).

 

An alkaline comet assay that was performed using test substance DTPMP (5-7Na) was read across to DTPMP (1-3Na). DTPMP (5-7 Na) salt (EC:701-216-4) was tested for genotoxicity in the in vivo Alkaline Comet Assay which was in accordance with OECD Test Guideline 489 and in compliance with GLP (Charles River Laboratories, 2022a, reliability 1). DTPMP (5 -7Na) salt was administered twice daily for two consecutive days via oral gavage to male Wistar rats at 500, 1000 and 2000 mg/kg bw/day in milli-Q water. No clinical signs of toxicity were observed. A positive control group was dosed twice by oral gavage with Ethyl Methane Sulfonate (EMS) at 200 mg /kg bw/day in physiological saline. Approximately 3-4 hours after the last dose the animals were sacrificed and single cell suspensions from liver, duodenum and stomach were made followed by Comet slide preparation. The slides were analysed and the Tail Intensity (%) was assessed. No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and stomach cells of test item treated animals compared to the vehicle (water) treated animals. In the stomach, mean Tail Intensities (%) were observed to be slightly higher than the 95% control limits of the distribution of the historical control data for the vehicle control (up to 12.63% versus 11.2%). However, the increase is minor and the results were within the maximum ranges observed in the historical data. Additionally, results were clearly distinct from the positive control (66.67%) and there was no statistically significant increase observed.

The mean Tail Intensity in liver, duodenum and stomach cells of vehicle-treated rats was 2.59 ± 0.52% (mean ± SD), 8.89 ± 0.68% (mean ± SD) and 11.34 ± 1.69% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control for liver and duodenum. The mean Tail Intensity in stomach cells of vehicle-treated rats was slightly above the 95% control limits (11.2%). However, this was considered to have no impact, as the increase is minor (0.14%), within the maximum range observed and it is clearly distinct from the positive control (66.67%). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 88.08 ± 2.64% (mean ± SD), 58.69 ± 2.75% (mean ± SD) and 66.67 ± 1.78% (mean ± SD) in male animals in liver, duodenum and stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analysed and the highest test dose was the maximum dose required by the guideline. Hence, all criteria for an acceptable assay were met. In conclusion, the test is valid and DTPMP (5-7 Na) salt is not genotoxic in the Comet assay in liver, duodenum and stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 2000 mg/kg (Charles River Laboratories, 2022, reliability 1).

 

 

DTPMP-H has been tested for clastogenicity in a valid in vivo chromosome aberration study in rat bone marrow following gavage with doses up to 1970 mg DTPMP-H/kg bw (Hazleton Laboratories, 1983). The study was conducted according to a protocol similar to OECD Test Guideline 475 with deviations and in compliance with GLP. The deviations included lower number of cells scored for aberrations and mitotic index than that required in the current guideline; the top dose resulted in 25% mortality and loss of body weight; metaphases were analysed from 4-6 animals per group; no evidence was presented for the test substance reaching the bone marrow. In the study the mitotic index was unaffected by the administration of the test substance and there was no evidence of clastogenicity was seen in rat bone marrow following a single oral gavage administration at doses up to the maximum tolerated dose of 1970 mg active acid/kg bw.

Justification for classification or non-classification

Based on the available data for DTPMP-H and DTPMP (5-7Na), no classification for genetic toxicity is required for DTPMP (1-3Na) according to Regulation (EC) No 1272/2008.