Sodium salts of [[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid (5-7 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): DTPMP (5-7Na) was 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: DTPMP (5-7Na) was positive in Chinese hamster lung CHL/IU cells (similar to OECD Test Guideline 473 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 one 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 C 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 in 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 analyzed	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 analyzed	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-dependant 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)

Deviations:

yes

Remarks:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemicals

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 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, 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 significant concentration-dependent increase

Statistics:

No statistical analysis performed

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

Positive controls validity:

valid

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

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

Positive controls validity:

valid

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

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)

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 L5178TK+/- 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 the solvent control cultures. The cytotoxicity of the test substance is assessed by post treatment cloning efficiency.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:

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 a valid in vivo mammalian alkaline comet assay study, conducted according to OECD Test Guideline 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.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Comet Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Apr 2021 to 08 Mar 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: The mean body weights were for males 268.3 ± 14.9 g and the range 225 – 290g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: Polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material equipped with water bottles. Up to 5 animals of the same sex and same dosing group were housed together.
- Diet (e.g. ad libitum): Pellets (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum, except during designated procedures. Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water. Freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 6 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 08/06/21 To: 22/07/2021

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: water (Milli-Q water)
- Justification for choice of vehicle: Product is aqueous solution hence preferred vehicle was water.
- Concentration of test material in vehicle: The test item was dissolved in water. Adjustment was made for specific gravity of the test
item (1.3108 g/mL) and the purity of the test item (24.93% w/w). Taking this into account 3.06 mL of pure test item was considered to be 1g of pure test item. Consequently to achieve for instance a 100 mg/mL solution, 3.06 mL of test item was added to 6.94 mL of vehicle.
- Amount of vehicle (if gavage or dermal): Dose volume: 10 mL/kg
- Type and concentration of dispersant aid (if powder): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Adjustment will be made for specific gravity of the test item (1.3108 g/mL) and the purity of the test item (24.93% w/w). Taking this into account 3.06 mL of pure test item is considered to be 1g of pure test item. The test item will be dissolved or suspended in water. Consequently to achieve for instance a 100 mg/mL solution, 3.06 mL of test item should be added to 6.94 mL of vehicle. Other concentrations can be calculated using this example.

Duration of treatment / exposure:

Two consecutive days

Frequency of treatment:

DRF Study: once daily
Main Study: Twice daily at 0 and 21 hours

Post exposure period:

Animals were sacrificed 3-4 hours after the second dose

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 - Vehicle (water) only

Dose / conc.:

500 mg/kg bw/day

Remarks:

Group 2 - Treatment group

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 3 - Treatment group

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Group 4 - Treatment group

No. of animals per sex per dose:

Main Study: 5 males per dose. Dose range finding study: one group of 3 male and 3 female animals.

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Ethyl Methane Sulphonate (EMS; CAS No. 62-50-0)
- Justification for choice of positive control(s): As per Table 1 of OECD Test Guideline 489
- Route of administration: oral gavage
- Doses / concentrations: 200 mg ethyl methane sulfonate (EMS)/kg body weight dissolved in physiological saline.

Tissues and cell types examined:

Single cell suspensions from liver, duodenum and stomach.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A dose range finding study was performed prior to this experiment where suitable doses were selected. In total 3 male and 3 female animals were dosed with the limit dose of 2000 mg/kg bw for 2 consecutive days. Clinical signs were recorded 2 hours and 21 hours after each dose. No mortalities occurred and no abnormalities were observed, therefore 2000 mg/kg bw was selected as the maximum dose in the main study (also the highest dose required in the current test guideline).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Treatment occurred during two consecutive days. Animals were sacrificed 3-4 hours after the second dose. Sampling occurred after termination of the animals.

Isolation of liver: The isolation method was based on the publication of Hu et al (2002). A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking water bath at 37°C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

Isolation of glandular stomach cells: This isolation method for glandular stomach is based on the JACVAM Comet validation study.
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free). The forestomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA). The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis. The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The glandular stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO was added immediately before use). The cell suspension was filtered through a 100 μm Cell Strainer to purify the cell suspension and collected in a tube and stored on ice.

Isolation of duodenum: This isolation method for duodenum is based on the JACVAM Comet validation study. The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA). The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodeunum was cut open and the surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded. The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use). The cell suspension was filtered through a 100 μm Cell Strainer to purify the cell suspension and collected in a tube and stored on ice.

DETAILS OF SLIDE PREPARATION: Slides were prepared immediately after the cell suspensions were obtained (finished within 3
hours after necropsy). To the cell suspension, melted low melting point agarose was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 μL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15-27 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

The cells on the slides were overnight (approximately 16-17 h) immersed in pre-chilled lysis solution in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution (1mM EDTA, pH 13) for 20 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1.0 Volt/cm (liver) or 0.7 Volt/cm (stomach and duodenum). The electrophoresis was performed for 20 (stomach and duodenum) or 30 (liver) minutes under constant cooling (actual temperature 4.0°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5-7 minutes. The slides were subsequently immersed for 5-6 minutes in Absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

METHOD OF ANALYSIS: To prevent bias, slides were randomly coded (per tissue) before examination of the comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system. One hundred fifty comets (50 comets of each replicate LMAgarose circle) were examined per sample. The following criteria for scoring of comets were used:

• Only horizontal orientated comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.

In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal. The occurrence of hedgehogs was scored in all treatment groups and the control. There were no hedgehogs present in any of the slides scored.

HISTOPATHOLOGY: Part of the liver, stomach and duodenum and the tail ID for identification from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was needed.

Evaluation criteria:

The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database.
c) Adequate numbers of cells and doses have been analysed.
d) The highest test dose is the MTD or 2000 mg/kg body weight/day
All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

A test item is considered clearly positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p <0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control, and
b) The increase is dose-related when evaluated with an appropriate trend test.
c) Any of the results are outside the 95% control limits of the historical negative control data range.

A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase at any sampling time when evaluated with an appropriate trend test,
c) All results are within the 95% control limits of the negative historical control data range
d) direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Remarks:

Water

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

EMS

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw/day for two consecutive days
- Solubility: Not specified
- Clinical signs of toxicity in test animals: In the dose-range finding test, three male and three female animals dosed with 2000 mg test item per kilogram body weight showed no treatment related clinical signs or mortality. No substantial differences in toxicity between sexes and so only males were used in the main study.
- Evidence of cytotoxicity in tissue analysed: Not applicable
- Rationale for exposure: maximum dose for the main test (the highest dose required in the current guideline)

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: The route of exposure and dose levels were appropriate according to the guideline. Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the maximum dose required by the guidelines. Hence, all criteria for an acceptable assay were met.
- Results and Statistical evaluation: 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 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.

Conclusions:

DTPMP (5-7 Na) salt (EC 701-216-4) was tested in a valid in vivo mammalian alkaline comet assay study, conducted according to OECD TG 489 and in compliance with GLP. 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

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-dependant 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-dependent 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).

 

DTPMP (5-7 Na) salt (EC 701-216-4) was tested for genotoxicity in the in vivo Alkaline Comet Assay 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 supporting in vivo chromosome aberration study in rat bone marrow following gavage with doses up to 1970 mg DTPMP-H/kg bw (Hazleton Laboratories, 1983, reliability score 2). 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, DTPMP (5-7Na), no classification for genetic toxicity is required for DTPMP (5-7Na) according to Regulation (EC) No 1272/2008.