Potassium phosphinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-22 till

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to the OECD guideline 476 and the EU Method B.17 performed in compliance to GLP standards

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium phosphinate

EC Number:

231-669-9

EC Name:

Sodium phosphinate

Cas Number:

7681-53-0

Molecular formula:

H3O2P.Na

IUPAC Name:

sodium phosphinate

Constituent 2

Reference substance name:

sodium hypophosphite

IUPAC Name:

sodium hypophosphite

Details on test material:

- Name of test material: Sodium hypophosphite; tested as monohydrate as the anhydrous form (CAS 7681-53-0) is highly hygroscopic and difficult to handle without specific precautions.
- Physical state: white crystalline solid (powder)
- Odour: none
- Supplier: Rhodia UK-Oldbury
- Batch number: 90113D
- Expiry date: June 2010
- Storage condition of test material: room temperature, stored in a tightly closed container in a dry and well-ventilated place, opening of the container restricted to the minimum needs, protected from humidity for eight days
- Disposal: incinerate the residues at an approved waste disposal site only
- Purity: 99.5%
- Impurities (identity and concentrations):
Phosphite (Na2HPO3): 0.14%
- Certificate of analysis:
Analysis number: ITC/09/01/04
Analysis date: 2009-01-27

No more data availabe

Method

Target gene:

Thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

78.13, 156.3, 312.5, 625, 1250 and 2500 µg/mL for both 3h and 24h exposure
Preliminary study: highest recommended dose-level of 5000 µg/mL induced an increase of osmolality in the culture medium of more than 50 mOsm/kg H2O, consequently the highest selected dose-level for the main test was 2500 µg/mL

Vehicle / solvent:

- Vehicle:
Test substance: culture medium (RPMI)
Positive controls: distilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: without S9 mix, 25 µg/mL (3h treatment) or 5 µg/mL (24h treatment)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: with S9 mix, 3 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: first experiment 3h with and without S9 mix; second experiment 3 h with S9 mix and 24h without S9 mix (as results of the first experiment were negative)
- Expression time (cells in growth medium): 48 hours (at 37°C in a humidified atmosphere of 5% CO2/95% air)

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: 2 cultures/dose-level and at least duplicate cultures for the controls

NUMBER OF CELLS EVALUATED: 2 x 10^5 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; relative suspension growth; cloning efficiency

No more data available

Evaluation criteria:

IWGT recommendations were followed and a positive result is considered when following criteria are fulfilled :
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control equals or exceeds the global evaluation factor (126 x 10E-6 for the microtiter method)
- a dose-related trend is demonstrated by a statistically significant trend test

Unless considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with RTG between 10 and 20%, is not considered as positive result.

A test item is determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adj. RTG
- there is no evidence of mutagenicity in a series of data points between 100 to 25% and there is also a negative data point between 10 and 1% Adj. RTG

Statistics:

No data

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality:Yes, at the dose-level of 5000 µg/mL the osmolality was equal to 391 mOsm/kg H2O (302 for the vehicle control). At dose-levels of 2500 and 1250 µg/mL the osmolality values were 348 and 320 mOsm/kg H2O respectively.
- Solubility: freely soluble in the vehicle at 100 mg/mL
- Precipitation: No precipitate was observed for the final dose-level of 5000 µg/mL using a treatment volume of 1000 µL/20mL
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA:
The values obtained in these experiments for the vehicle and positive control are in agreemant with the historical data (see overall remarks)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The cloning efficiencies CE₂ and the mutation frequencies of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered valid.

 

Since the test item was freely soluble, non-toxic in the preliminary test and no precipitate was observed at the highest dose-level of 5000 µg/mL, the highest dose-level was set at this value.

Cytotoxicity and mutagenicity results

- Experiments without S9 mix (Table 1: first experiment, 3h treatment and table 2: second experiment, 24h treatment)

No noteworthy cytotoxicity was induced in either experiment, since no noteworthy decrease in the Adj. RTG was observed at any dose-level.

No noteworthy increase in the mutation frequency was noted at any dose-level.

- Experiments with S9 mix (Table 3: first experiment, 3h treatment and table 4: second experiment, 3h treatment)

No noteworthy cytotoxicity was induced in either experiment, since no noteworthy decrease in the Adj. RTG was observed at any dose-level.

No noteworthy increase in the mutation frequency was noted at any dose-level.

Table 1: Cytotoxicity and mutagenicity results of the first experiment without S9 mix, 3h treatment

Doses µg/mL		Cytotoxicity			Mutagenicity
		Adj. RTG %	Adj. RSG %	RCE2 %	MF x10^-6	R
0	C1	100	100	100	144	1.0
	C2	100	100	100	219	1.0
78.13	C1	89	110	81	190	1.3
	C2	122	92	132	164	0.8
156.3	C1	90	91	99	217	1.5
	C2	135	109	124	145	0.7
312.5	C1	85	95	89	177	1.2
	C2	125	107	117	160	0.7
625	C1	65	161	41	225	1.6
	C2	133	133	100	170	0.8
1250	C1	107	106	101	129	0.9
	C2	150	128	117	165	0.8
2500	C1	72	86	83	152	1.1
	C2	125	125	100	178	0.8
MMS 25 µg/mL	C1	30	48	62	788	5.5
	C2	46	76	60	857	3.9

Adj. RTG: Adjusted relative total growth

Adj. RSG: Adjusted relative suspension growth

RCE₂: relative cloning efficiency

MF: Mutation frequency

R: Ration between mutation frequency of treated cells/mutation frequency of control cells

 

Table 2: Cytotoxicity and mutagenicity results of the second experiment without S9 mix, 24h treatment

Doses µg/mL		Cytotoxicity			Mutagenicity
		Adj. RTG %	Adj. RSG %	RCE2 %	MF x10^-6	R
0	C1	100	100	100	96	1.0
	C2	100	100	100	77	1.0
78.13	C1	135	119	114	60	0.6
	C2	112	85	132	95	1.2
156.3	C1	116	94	124	58	0.6
	C2	102	98	104	86	1.1
312.5	C1	134	93	144	44	0.5
	C2	119	105	114	71	0.9
625	C1	128	96	133	46	0.5
	C2	122	115	106	53	0.7
1250	C1	103	109	94	76	0.8
	C2	94	98	96	80	1.0
2500	C1	112	89	126	65	0.7
	C2	127	102	124	59	0.8
MMS 25 µg/mL	C1	75	74	101	453	4.7
	C2	72	93	77	440	5.7

Adj. RTG: Adjusted relative total growth

Adj. RSG: Adjusted relative suspension growth

RCE₂: relative cloning efficiency

MF: Mutation frequency

R: Ration between mutation frequency of treated cells/mutation frequency of control cells

 

Table 3: Cytotoxicity and mutagenicity results of the first experiment with S9 mix, 3h treatment

Doses µg/mL		Cytotoxicity			Mutagenicity
		Adj. RTG %	Adj. RSG %	RCE2 %	MF x10^-6	R
0	C1	100	100	100	175	1.0
	C2	100	100	100	173	1.0
78.13	C1	102	76	134	166	0.9
	C2	112	123	92	161	0.9
156.3	C1	109	83	132	116	0.7
	C2	88	97	90	191	1.1
312.5	C1	107	85	126	223	1.3
	C2	99	132	75	202	1.2
625	C1	115	102	112	154	0.9
	C2	104	105	99	204	1.2
1250	C1	116	82	142	177	1.0
	C2	109	78	139	131	0.8
2500	C1	121	92	132	150	0.9
	C2	96	100	96	190	1.1
MMS 25 µg/mL	C1	40	70	57	955	5.5
	C2	31	62	50	1115	6.5

Adj. RTG: Adjusted relative total growth

Adj. RSG: Adjusted relative suspension growth

RCE₂: relative cloning efficiency

MF: Mutation frequency

R: Ration between mutation frequency of treated cells/mutation frequency of control cells

 

Table 4: Cytotoxicity and mutagenicity results of the second experiment with S9 mix, 3h treatment

Doses µg/mL		Cytotoxicity			Mutagenicity
		Adj. RTG %	Adj. RSG %	RCE2 %	MF x10^-6	R
0	C1	100	100	100	67	1.0
	C2	100	100	100	72	1.0
78.13	C1	79	56	142	78	1.2
	C2	89	89	100	85	1.2
156.3	C1	115	73	157	59	0.9
	C2	96	68	140	78	1.1
312.5	C1	97	58	166	99	1.5
	C2	104	102	101	66	0.9
625	C1	89	63	142	61	0.9
	C2	84	67	124	68	1.0
1250	C1	108	95	114	57	0.9
	C2	111	114	97	52	0.7
2500	C1	87	58	149	70	1.1
	C2	95	78	121	76	1.1
MMS 25 µg/mL	C1	12	37	32	1551	23.2
	C2	9	36	24	1830	25.6

Adj. RTG: Adjusted relative total growth

Adj. RSG: Adjusted relative suspension growth

RCE₂: relative cloning efficiency

MF: Mutation frequency

R: Ration between mutation frequency of treated cells/mutation frequency of control cells 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under these experimental conditions, no noteworthy increase in the mutation frequency was observed, both with and without S9 mix. Sodium hypophosphite did not show any mutagenic activity in the in vitro mammalian cell gene mutation test with L5178Y TK+/- mouse lymphoma cells.

Executive summary:

The potential of sodium hypophosphite to induce mutations in L5178Y TK+/- mouse lymphoma cells was assessed according to OECD guideline 476 and EU method B.17). The study was conducted in compliance with the principles of Good Laboratory Practice regulations on the monohydrate form of the test item as the anhydrous form is highly hygroscopic and difficult to handle without specific precautions.

A preliminary toxicity test was performed to define the dose-levels of sodium hypophosphite to be used for the mutagenicity study. The test item was

tested in two independant experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction of rats induced with Aroclor 1254. Vehicle controls and positive controls were included in both experiments.

Mouse lymphoma L5178Y TK+/- cells were exposed to the following dose-levels of sodium hypophosphate (two cultures/dose-level) for both experiments: 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/mL. The treatment duration of the first experiment was 3 hours with and without S9 mix and that of the second experiment was 3 hours with S9 mix and 24 hours without S9 mix (as the results of the first experiment were negative). After an expression time of 48 hours at 37°C in a humidified atmosphere of 5% CO2/95% air, trifluorothymidine was used as selection agent. The evaluation of toxicity was performed on the basis of decreasing values for adjusted relative total growth (Adj. RTG), relative suspension growth (Adj. RSG) and cloning efficiency (CE₂) and/or increase in mutation frequency.

The cloning efficiencies (CE₂₎ and the mutation frequencies for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered valid.

No toxicity was noted in these experiments, both with and without S9 mix. The test item did not induce any significant increase in the mutation frequency, both with and without S9 mix.

Under these experimental conditions sodium hypophosphite did not show any mutagenic activity in the in vitro mammalian cell gene mutation test

using mouse lymphoma cells.