Reaction mass of trisodium 2-(hydroxyphosphinato)succinate and pentasodium 1-(hydroxyphosphinato)butane-1,2,3,4-tetracarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 9 september 1992 To 15 october 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity of the test material is based on the phosphonate composition, with no direct measure of % sodium. ITC 288 is not the "Reaction mass of tetrasodium-phosphonoethane-1,2-dicarboxylate and hexasodium-phosphonobutane-1,2,3,4--tetracarboxylate" (EC 410-800-5) rather the substance "Reaction mass of trisodium-phosphonoethane-1,2-dicarboxylate and pentasodium-phosphonobutane-1,2,3,4--tetracarboxylate" (EC 701-079-0) as demonstrated by the manufacturing specs reported in Section 1.2, legal entity composition.

Target gene:

Histidine gene (S. Typhimurium)
Tryptophane gene (E. Coli (WP2uvrA-))

Species / strain / cell type:

bacteria, other: Salmonella typhimurium: TA1535, TA1537, TA98, and TA100. Escherichia coli: WP2uvrA-

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 from induced Aroclor rat liver

Test concentrations with justification for top dose:

0, 8, 40, 200, 1000, and 5000 µg/plate (experiment 1)
0, 312.5, 625, 1250, 2500 and 5000 µg/plate (experiment 2)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9: N-Ethyl-N'-nitro-N-nitrosoguanidine 3 µg/plate for TA100 and 5 µg/plate for TA1535, 9-aminoacridine 80 µg/plate for TA1537,4-Nito-o-phenylenediamine 5 µg/plate for TA 1538 and 4-Nitroquinoline-1-oxide 0.2 µg/plate for TA98. + S9: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48 h at 37 °C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method:
The cytotoxicity of the test material was determined using TA 100 or WP2 uvrA-. Five doses(0, 8, 40, 200, 1000, and 5000 µg/plate ) of ITC 288 were tested in duplicate. After 48 hours incubation at 37°C the plates were scored for revertant colonies and examined for a thinning of the background lawn.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate.

Statistics:

No data

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

none

Remarks on result:

other: all strains/cell types tested

Table 7.6.1/1:Number of revertants per plate (mean of 3 plates), (TA 100, TA 1535 and WP2uvrA- +/- S9 in experiment 1)

	[Strain TA 100]			[Strain TA 1535]			WP2uvrA-
Conc. [µg/plate]	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity
0*	170	186.3	no	17	17.7	no	17.7	18.3	no
8.0	156.7	162.0	no	16.3	20.0	no	16.3	18.7	no
40	167.7	153.7	no	18.7	15.0	no	10.3	14.0	no
200	151.7	149.0	no	17.7	17.3	no	17.7	14.0	no
1000	165.7	144.0	no	21.3	15.7	no	11.3	13.3	no
5000	170.7	168.7	no	14.7	13.0	no	15	14.7	no
Positive control	695	421.7	no	348.7	306.7	no	447.7	187.0	no

*solvent control with water.

Table 7.6.1/2:Number of revertants per plate (mean of 3 plates), (TA98 and TA 1537 +/- S9 in experiment 1)

	[Strain TA 98]			[Strain TA 1537]
Conc. [µg/plate]	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity
0*	20	28	no	14.3	11.7	no
8.0	18.3	23.7	no	9.0	14.7	no
40	21.7	23.3	no	13.7	10.3	no
200	20.3	26.7	no	12.3	11.3	no
1000	16.7	31.0	no	9.7	11.7	no
5000	16.3	21.3	no	13.0	12.0	no
Positive control	183.3	263.3	no	454	144.0	no

*solvent control with water.

Table 7.6.1/3:Number of revertants per plate (mean of 3 plates), (TA 100, TA 1535 and WP2uvrA- +/- S9 in experiment 2)

	[Strain TA 100]			[Strain TA 1535]			WP2uvrA-
Conc. [µg/plate]	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity
0*	160.0	174.7	no	11.0	12.0	no	16	11.7	no
312.5	154.7	141.7	no	14.0	9.5	no	13.7	11.3	no
625	127.3	159.0	no	14.3	9.0	no	11.0	15.3	no
1250	163.5	148.0	no	13.0	11.7	no	13.0	15.7	no
2500	124.3	169.3	no	14.0	13.3	no	15.7	11.7	no
5000	158.3	136.7	no	11.0	12.3	no	13.0	15.0	no
Positive control	508.3	436.3	no	161.3	142	no	599.0	111.3	no

*solvent control with water.

Table 7.6.1/4:Number of revertants per plate (mean of 3 plates), (TA98 and TA 1537 +/- S9 in experiment 2)

	[Strain TA 98]			[Strain TA 1537]
Conc. [µg/plate]	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity
0*	17.3	22.3	no	6.0	9.3	no
312.5	22.3	20.0	no	8.7	6.7	no
625	25.0	19.7	no	10.7	13.0	no
1250	22.0	17.7	no	7.7	4.0	no
2500	22.0	22.7	no	8.0	8.3	no
5000	20	17.7	no	8.7	7.7	no
Positive control	203.7	158.0	no	301	161.0	no

*solvent control with water.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the conditions of this test, ITC 288 was found to be non-mutagenic in Bacteria.

Executive summary:

In a reverse gene mutation assay in bacteria (Thompson, 1992), Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with ITC 288 by the Ames plate incorporation method at five dose levels, in triplicate, both with and without S9. The dose range determined in a preliminary toxicity assay was 8 to 5000 µg/plate using TA100 or WP2 uvrA-. In the mutation study, the dose range used were 8 to 5000 µg/plate in the first experiment and 312.5 to 5000 µg/plate in the second experiment. The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. ITC 288 caused no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of salmonella and escherichia used with and without S9. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of ITC 288, either with or without metabolic activation (experiment 1 + experiment 2). Under the conditions of this test, ITC 288 was found to be non-mutagenic in Bacteria.

This study is classified as acceptable and satisfies the requirement for test Guideline OECD 471 for in vitro mutagenesis (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 July 1996 To 23 May 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing: Report, Part II revised (Supplementary Mutagenicity Tests: UKEMS recommended procedures, 1993)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

Purity of the test material is based on the phosphonate composition, with no direct measure of % sodium. ITC 288 is not the "Reaction mass of tetrasodium-phosphonoethane-1,2-dicarboxylate and hexasodium-phosphonobutane-1,2,3,4--tetracarboxylate" (EC 410-800-5) rather the substance "Reaction mass of trisodium-phosphonoethane-1,2-dicarboxylate and pentasodium-phosphonobutane-1,2,3,4--tetracarboxylate" (EC 701-079-0) as demonstrated by the manufacturing specs reported in Section 1.2, legal entity composition.

Species:

rat

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Margate, Kent.
- Age at study initiation: 6-9 weeks old
- Weight at study initiation: 221-265 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: up to 3 in solid-floor polypropylene cages
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data
- Amount of vehicle (if gavage or dermal): 10 ml/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was freshly prepared as required as a solution at the appropriate concentrations in distilled water

Duration of treatment / exposure:

not applicable

Frequency of treatment:

a single oral dose

Post exposure period:

2 hours (experiment 2) and 16 hours (experiment 1)

Remarks:

Doses / Concentrations:
700 and 2000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

4 males per dose (test substance and positive control) + 6 males (vehicle control)

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene (AAF) and N,N'-Dimethylhydrazine dihydrochloride (NDHC) were freshly prepared as required as a suspension at the appropriate concentration in arachis oil BP and in sterile distilled water respectively.
- Route of administration: oral
- Doses / concentrations: 50 mg/kg (AAF) and 20 mg/kg (NDHC)

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range-finding toxicity study was performed to determine a suitable dose level for the main UDS study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In the first experiment, two groups of 4 rats were dosed with test material at 700 and 2000 mg/kg. In addition, two further groups of rats were included in the experiment, one group (6 rats) was dosed with the vehicle alone (distilled water) and the second group (4 rats) was dosed with Acetylaminofluorine as a positive controls. All animals were perfused approximately 16 hours after dosing. For pratical reasons the whole experiment was performed in phases, with animals from each group treated in each phase. In the second experiment, the dose groups and group sizes were as for Experiment 1 except that the positive control was N,N'-dimethylhydrazine dihydrochloride and animals were perfused approximately 2 hours after dosing.

DETAILS OF SLIDE PREPARATION:
The cells were fixed in freshly prepared 1:3 acetic acid:methanol, three changes of fixative were used. Finally cells were washed with distilled water and the coverslips allowed to air dry and then mounted onto the ends of glass slides, cell side up, and left to dry overnight in a dust free environment. The coverslips were coated with Ilford K2 autoradiographic emulsion and incubated at 4°C for 7 to 14 days in a sealed light proof container. Following the exposure period the slides were processed using photographic developer and fixative, and then the cells were stained using haematoxylin/eosin. When the cells were dry they were coverslipped using a mounting medium. The slides were then assessed for obvious signs of toxicity, reduced numbers of cells and poor labelling.

METHOD OF ANALYSIS:
The coded slides were scored using an automated image analysis system linked to a computer program (Grain). The method used to score the slides was an area counting method. A minimum of three slides for each animal were scored with a maximum of 50 cells per slide giving an accumulative total or 150 cells per animal. The number of silver grains within the nucleus were first observed and recorded as the Nuclear Count (N). Three cytoplasmic areas (each equal to the nuclear area) were also scored to give the Mean Cytplasmic Count (C). A net Nuclear Grain Count (N-C), was then calculated. Cells in 'S'-phase were not scored for grain counts, these were easily recognisable due to the dense 'graining' appearance of the nucleus. Mean values of (N), (C), (N-C) and percentage cells in repair (%R)were calculated. Values of (N-C) are typically in the range of -6 to -2 for vehicle controls, although variations in experimental conditions can give values outside of this range.

Evaluation criteria:

In positive responses at least 20% of all cells assessed should be in repair, ie. undergoing unscheduled DNA synthesis, having a net Nuclear Grain (N-C) value of +5 or greater.

Statistics:

No statistics were performed.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

- Range-finding study: No clinical signs were observed in animals at 1000 and 2000 mg/kg and there were no premature deaths. - UDS study (experiment 1+ experiment 2): no clinical signs and no premature deaths were observed.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: none


RESULTS OF DEFINITIVE STUDY
There were no premature deaths seen in any of the dose groups, and no clinical signs were observed in any animals in either of the two experiments.

Any other information was available.

Conclusions:

Interpretation of results: negative
Under the test conditions, ITC 288/S did not induced an increase in the incidence of cells undergoing unscheduled DNA synthesis in isolated rat hepatocytes following in vivo exposure for 2 and 16 hours. Therefore, ITC 288/S was considered to be non-genotoxic.

Executive summary:

In an in vivo liver unscheduled DNA synthesis assay (Durward, 1997), ITC 288/S was administered to male rats at the maximum recommended dose of 2000 mg/kg with 700 mg/kg as the lower dose level. The study was performed in two parts, in experiment 1 the livers were perfused approximately 16 hours after dosing and, in experiment 2, perfusion was performed approximately 2 hours after dosing. Following perfusion the liver hepatocytes were processed to give stained slides which were then scored using a microscope and an automated image analysis system. Further groups of rats were given a single oral dose of distilled water, acetylaminofluorene (AAF) at 16 hours or N,N'-dimethyl hydrazine dihydrochloride (NDHC) at 2 hours to serve as vehicle and positive controls respectively. There was no increase in the incidence of unscheduled DNA synthesis in animals dosed with test material at either time point. The positive controls both produced marked increases in the incidence of cells in repair. Under the conditions of this test, ITC 288/S was considered to be non-genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro and in vivo, the following genotoxic endpoints were assessed:

- Gene mutation potential: one reverse bacterial gene mutation test (Thompson #1, 1992) was selected as a key study, (OECD 471, Kr: 1). In this study, ITC 288/S is not mutagenic in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA- with and without metabolic activation, up to a limit concentration. These results are confirmed by a supporting study (Thompson #2, 1992), (OECD 471, Kr: 2) providing negative results with or without metabolic activation. Therefore, ITC 288/S showed no mutagenic action in Bacteria.

- Chromosomal aberration potential: In vitro, one cytogenetic study in CHO cells (OECD 473, Kr: 1) was available and was selected as the key study (Wright, 1993). The results were negative with metabolic activation, up to limit concentration. Weak clastogenic effects were observed at the maximum concentration used without metabolic activation, together with a high level of cytotoxicity (cell survival reduced by 80%) and at the medium concentration (7.5% of cells with aberrations), but based on "Evaluation criteria" of this test , these results were considered as ambiguous. In vivo, ITC 288/S was also evaluated for its ability to induce chromosomal aberrations in a mouse micronucleus test (Durward R, 1993), (key study, OECD 474, Kr:1). No increase in micronucleated polychromatic erythrocytes (micronuclei) and no significant change in the NCE/PCE ratio was observed in male or female mice 24,48 or 72 hours following i.p. injection of 1000 mg/kg. Therefore, ITC 288/S is considered as not genotoxic.

- DNA repair potential:

In the in vivo liver unscheduled DNA synthesis (UDS) assay,(Durward, 1997), (key study, OECD 486, Kr:1), male rats were treated by gavage with ITC 288/S at 700 and 2000 mg/kg body weight. Primary hepatocytes cultures were prepared 2 and 16 hours later, incubated with 3H-thymidine and assessed for induction of UDS. No DNA repair activity was seen, therefore ITC 288/S is considered as not genotoxic.

In conclusion: based on all these studies, ITC 288/S is considered as not genotoxic.

Short description of key information:
- Mutagenicity: negative in Bacteria (OECD 471, Kr:1).
- Clastogenicity/Aneugenicity: ambiguous in vitro and negative in vivo (OECD 473 and OECD 474, Krs:1).
- DNA damage and/or repair: negative in vivo (OECD 486, Kr:1).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro and in vivo data, ITC 288/S is considered as not genotoxic, therefore no classification is required according to the EU legislation (Directive 67/548/EEC and CLP regulation (1272/2008).