Potassium phosphinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There were no data for Potassium phosphinate regarding its genotoxic potential however the genetic toxicity of sodium hypophosphite was assessed in 3 in vitro studies conducted in compliance with the principles of Good Laboratory Practices:
- a gene mutation assay on Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 (OECD guideline 471)
- an in vitro chromosomal aberration assay in cultured human lymphocytes (OECD guideline 473)
- an in vitro gene mutation assay in mouse lymphoma L5178Y cells (OECD guideline 476)
In all three studies negative results were reported in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

Thymidine kinase locus

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

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)

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

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'.

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 

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.

Reference 2

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:

2009-05-27 till 2009-07-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to the OECD guideline in compliance to GLP standards

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S.typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (liver post mitochondrial fraction of rats induced with Aroclor 1254)

Test concentrations with justification for top dose:

- First mutagenicity experiment with and without S9 mix: 312.5, 625, 1250, 2500 and 5000 µg/plate
- Second mutagenicity experiment with and without S9 mix: 625, 1250, 2500, 3750 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: water (for injections)
- Justification for choice of solvent/vehicle: highly soluble in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- In agar (plate incorporation): first mutagenicity test and second mutagenicity test without S9 mix
- Preincubation (60 minutes at 37°C ) only in second mutagenicity test with S9 mix
- Exposure duration: 48h to 72h

SELECTION AGENT (mutation assays): agar containing traces of histidine and biotin, maintained at 45°C

NUMBER OF REPLICATIONS: two independent mutagenicity experiments each using three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: the evaluation of the toxicity was based on the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.
Reference to historical data or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

No statistical analysis performed

Species / strain:

other: S.typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to limit concentrations recommended by the test guideline

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: freely soluble at 50 mg/mL
- Precipitation: No presipitate observed
- Other confounding effects: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

- The number of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

 

- Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

 

- No precipitate was observed in the petri plates when scoring the revertants at any of the tested dose-levels.

 

- No toxicity was noted at any dose-level in any strain.

 

- A noteworthy increase in the number of revertants was noted in the TA 1537 strain (up to 3.1-fold the vehicle control value) in the second experiment without S9 mix. This increase exceeded the threshold of 3-fold the vehicle control value, but it was not reproducible since it was not observed in the first experiment performed under the same experimental conditions. Moreover, the corresponding mean revertant colony counts remained within the historical data range for the vehicle control (11 versus 3-13 for the historical data). Consequently, this increase was not considered as biologically relevant.

 

- A slight increase in the number of revertants was noted in the TA 100 strain (up to 1.8-fold the vehicle control value) in the second experiment with S9 mix (preincubation method). Since this increase did not reach the threshold of 2-fold the vehicle control value and was neither observed in the first experiment (direct plate incorporation method) nor clearly dose-related, it was not considered as biologically relevant.

 

- The test item did not induce any other noteworthy increase in the number of revertants, in any of the other strains, either with or without S9 mix,.

- Table: Detailed results for the two mutagenicity test performed with five bacterial strains for five concentrations of the test substance sodium hypophosphite as well as the vehicle and positive control.

Strain	Compound	First mutagenicity test				Second mutagenicity test
		Dose level (µg/plate)	S9 mix	Revertant colony count	Revertant colony count	Dose level (µg/plate)	S9 mix	Revertant colony count	Revertant colony count
				Mean	SD			Mean	SD
TA 1535	Water for injection		without	13	5		without	29	8
	Sodium hypophosphite	312.5	without	15	2	625	without	26	8
	Sodium hypophosphite	625	without	15	5	1250	without	37	3
	Sodium hypophosphite	1250	without	16	11	2500	without	30	8
	Sodium hypophosphite	2500	without	13	6	3750	without	30	3
	Sodium hypophosphite	5000	without	15	8	5000	without	33	14
	Sodium azide	1	without	768	54	1	without	672	15
	Water for injection		with	16	5		with	25	10
	Sodium hypophosphite	312.5	with	12	2	625	with	19	3
	Sodium hypophosphite	625	with	14	4	1250	with	15	2
	Sodium hypophosphite	1250	with	16	8	2500	with	30	11
	Sodium hypophosphite	2500	with	23	6	3750	with	20	6
	Sodium hypophosphite	5000	with	13	1	5000	with	21	7
	2-Anthramine	2	with	184	22	2	with	87	7
TA 1537	Water for injection		without	5	1		without	4	3
	Sodium hypophosphite	312.5	without	9	4	625	without	6	4
	Sodium hypophosphite	625	without	13	2	1250	without	7	1
	Sodium hypophosphite	1250	without	7	1	2500	without	8	2
	Sodium hypophosphite	2500	without	12	4	3750	without	8	3
	Sodium hypophosphite	5000	without	8	4	5000	without	11	1
	9-Aminoacridine	50	without	194	15	50	without	749	137
	Water for injection		with	12	7		with	10	1
	Sodium hypophosphite	312.5	with	8	2	625	with	14	7
	Sodium hypophosphite	625	with	5	1	1250	with	15	7
	Sodium hypophosphite	1250	with	13	8	2500	with	12	6
	Sodium hypophosphite	2500	with	8	9	3750	with	7	1
	Sodium hypophosphite	5000	with	21	6	5000	with	10	4
	2-Anthramine	2	with	111	6	2	with	56	6
TA 98	Water for injection		without	30	6		without	26	4
	Sodium hypophosphite	312.5	without	19	6	625	without	27	4
	Sodium hypophosphite	625	without	22	3	1250	without	20	6
	Sodium hypophosphite	1250	without	24	20	2500	without	27	4
	Sodium hypophosphite	2500	without	26	3	3750	without	29	3
	Sodium hypophosphite	5000	without	30	1	5000	without	21	4
	2-Nitrofluorene	0.5	without	161	5	0.5	without	165	19
	Water for injection		with	35	5		with	25	8
	Sodium hypophosphite	312.5	with	26	6	625	with	35	9
	Sodium hypophosphite	625	with	39	5	1250	with	37	3
	Sodium hypophosphite	1250	with	41	1	2500	with	39	4
	Sodium hypophosphite	2500	with	37	6	3750	with	30	15
	Sodium hypophosphite	5000	with	40	6	5000	with	32	8
	2-Anthramine	2	with	741	38	2	with	872	148
TA 100	Water for injection		without	124	8		without	136	11
	Sodium hypophosphite	312.5	without	149	15	625	without	153	11
	Sodium hypophosphite	625	without	148	8	1250	without	150	37
	Sodium hypophosphite	1250	without	154	15	2500	without	156	20
	Sodium hypophosphite	2500	without	155	13	3750	without	160	39
	Sodium hypophosphite	5000	without	145	14	5000	without	149	18
	Sodium azide	1	without	698	110	1	without	837	141
	Water for injection		with	147	8		with	92	9
	Sodium hypophosphite	312.5	with	139	14	625	with	101	27
	Sodium hypophosphite	625	with	145	36	1250	with	134	21
	Sodium hypophosphite	1250	with	162	20	2500	with	135	30
	Sodium hypophosphite	2500	with	163	25	3750	with	131	8
	Sodium hypophosphite	5000	with	169	9	5000	with	167	17
	Benzo(a)pyrene	5	with	353	16	5	with	563	72
TA 102	Water for injection		without	372	18		without	374	39
	Sodium hypophosphite	312.5	without	353	26	625	without	393	26
	Sodium hypophosphite	625	without	347	32	1250	without	418	11
	Sodium hypophosphite	1250	without	428	41	2500	without	451	21
	Sodium hypophosphite	2500	without	386	20	3750	without	340	8
	Sodium hypophosphite	5000	without	397	39	5000	without	389	18
	Mitomycin C	0.5	without	3041	95	0.5	without	2081	158
	Water for injection		with	582	40		with	557	26
	Sodium hypophosphite	312.5	with	566	77	625	with	469	29
	Sodium hypophosphite	625	with	458	15	1250	with	528	105
	Sodium hypophosphite	1250	with	470	24	2500	with	475	46
	Sodium hypophosphite	2500	with	512	70	3750	with	548	95
	Sodium hypophosphite	5000	with	586	31	5000	with	558	106
	2-Anthramine	10	with	2775	29	10	with	1941	136

Conclusions:

Interpretation of results (migrated information):
negative

Under these experimental conditions, no noteworthy increase in the number of revertants was observed towards all the strains used, both with and without S9 mix. Sodium hypophosphite did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The potential of sodium hypophosphite to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium according to the OECD guideline 471 and the EU Method B13/14. The study was conducted in compliance with the principles of Good Laboratory Practice on the monohydrated 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 then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

 

The five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed to the following dose-levels of sodium hypophosphite (three plates/dose-level):

- 312.5, 625, 1250, 2500 and 5000 µg/plate, for the first mutagenicity experiment with and without S9 mix,

- 625, 1250, 2500, 3750 and 5000 µg/mL for the second mutagenicity experiment with and without S9 mix.

After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

No precipitate was observed in the petri plates when scoring the revertants at all dose-levels.

No toxicity was noted towards all the strains used, both with and without S9 mix.

The test item did not induce any significant increase in the number of revertants, both with or without S9 mix, in any of the five strains.

Under these experimental conditions sodium hypophosphite did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-05-27 till 2009-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to the OECD guideline 473 and the EU Method B.10, performed in compliance to GLP standards

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Origin: primary cell cultures prepared from whole blood samples from two healthy donors
- Type and identity of media: RPMI 1640 medium containing 20% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and phytohemagglutinin (mitogen)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (liver post-mitochondrial fraction and cofactors of rats induced with Aroclor 1254)

Test concentrations with justification for top dose:

- First experiment (with and without S9 mix)
Mitotic index: 19.53, 39.06, 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/mL
Chromosome aberration (metaphase analysis): 625, 1250 and 2500 µg/mL

- Second experiment (with and without S9 mix)
Mitotic index: 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL
Chromosome aberration (metaphase analysis): 625, 1250 and 2500 µg/mL for 20h treatments and 2500 µg/mL for 44h treatments

Vehicle / solvent:

- Vehicle used:
Test substance: culture medium (RPMI)
Positive controls: water (for injections)

No more data available

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: without S9 mix, 3 µg/mL (3h of treatment) or 0.2 µg/mL (continuous treatment)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: with S9 mix, 12.5 and 25 µg/mL for mitotic index and only 12.5 µg/mL for chromosome aberration

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48h (at 37°C)
- Exposure duration:
First experiment: 3h
Second experiment: without S9 mix continuously until harvesting; with S9mix 3h
- Fixation time (start of exposure up to harvest of cells):
First experiment: 20h (approximately 1.5 normal cell cycles)
Second experiment: 20h and 44h (respectively 1.5 normal cell cycles and 24 hours later)

SPINDLE INHIBITOR (cytogenetic assays): colcemid solution (10 µg/mL), treatment one and a half hours before harvest
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED:
Cytotoxicity: 1000 cells evaluated
Chromosome aberration: 200 metaphases in total (i.e. 100 metaphases/culture). When at least 10% cells with structural chromosome aberration were observed only 50 metaphases/culture were evaluated

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

OTHER:
- Cell culture preparation: 0.5 mL of heparinized whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, 2mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and phytohemagglutinin (mitogen)
- Preparation of microscope slides: after treatment with spindle inhibitor each culture recieved a hypotonic treatment (KCl 0.075 M). Afterwards cells were fixed in a methanol/acetic acid mixture (3/1; v/v) and spread on glass slides before staining
- All metaphase analyses were performed blind
- Structural aberrations recorded: gaps, chromatid and chromosome breaks and exchanges, multiple aberrations and pulverizations

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

The frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, these results were compared using a chi-square test in which p = 0.05 was used as the lowest level of significance

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up the maximal practicable concentration regarding osmolality of the culture medium.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: Yes, osmolality was 302 mOsm/kg H2O in vehicle control, while in dose-level of 5000 µg/mL it was 378 mOsm/kg H2O. Consequently, this dose-level was not chosen as highest dose-level since it induced an increase in osmolality greater than 50 mOsm/kg H2O. The dose-level of 2500 µg/mL had an osmolality equal to 342 mOsm/kg H2O, thus equivalent to the vehicle control and was thus chosen as highest dose level.
- Solubility in
vehicle: freely soluble at 55 mg/mL
- Precipitation: no precipitate in the culture medium at the highest dose-level of 5000 µg/mL
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA:
The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was within the range provided by historical data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiments without S9 mix: No noteworthy decrease in the mitotic index was noted at any of the tested dose-levels following the 3, 20 or the 44 hour treatments
- Experiments with S9 mix: In the first experiment a slight to moderate decrease in mitotic index was observed at dose levels =156.3µg/mL (37 to 58% decrease). In the second experiment, no noteworthy decrease in mitotic index was noted at any of the tested dose-levels, at any of the harvest times.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

- Frequency of cells with structural chromosome aberrations for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

- Metaphase analysis

For both experiments with and without S9 mix no significant increase in the frequency of cells with structural chromosomal aberrations (such as chromatid-type or chromosome-type deletions or exchanges) was noted for either of the 3, 20 or the 44 hour treatments and for either of the harvest times.

- Results of the first experiment with and without S9 mix

The first experiment exposing cells to the test substance with and without metabolic activation for 3h and harvested at a time equivalent to about 1.5 normal cell cycle (20h) gave negative results

- Results of the second experiment without S9 mix (20h continuous treatment)

As prescribed by the guidelines an additional experiment needed to be performed as the first experiment gave negative results under all tested conditions.

The second experiment was therefore performed without activation with continuous treatment until harvesting at a time equivalent to about 1.5 normal cell cycle lengths.

Structural chromosome aberrations scored: gaps, chromatid and chromosome deletions and exchanges, multiple aberrations and pulverizations.

Doses (µg/mL)	Slide n°	Mitotic index (%)	Mitotic index mean (%)		# cells scored	Numerical aberrations	Structural chromosome aberrations		Cells with structural chromosome aberrations
							total with gaps	total without gaps	total with gaps	mean (%)	total without gaps	mean (%)
0; vehicle control	43 C1	5.3	5.00		100	0	5	5	3	2.5	3	2.5
	60 C2	4.7			100	0			2		2
78.1	70 C1	7.2	5.95
	69 C2	4.7
156.3	71 C1	4.7	4.25
	44 C2	3.8
312.5	59 C1	5.5	5.60
	48 C2	5.7
625	74 C1	3.9	5.00		100	0	5	5	1	2.5	1	2.5
	68 C2	6.1			100	1			4		4
1250	47 C1	3.2	4.55		100	0	3	3	0	1.0	0	1.0
	67 C2	5.9			100	0			2		2
2500	58 C1	3.1	3.85		100	1	4	2	3	2.0	2	1
	51 C2	4.6			100	0			1		0
0.2; Mitomycin C	65 C1	3.3	3.10		50	0	34	34	9	25.0	9	25.0
	63 C2	2.9			50	0			16		16

C1: culture 1

C2: culture 2

Conclusions:

Interpretation of results (migrated information):
negative

Under these experimental conditions, no statistically significant increase in the frequency of cells with chromosome aberrations was observed, both with and without S9 mix, using a dose range from 19.53 to 2500 µg/ml (maximal practicable concentration regarding the osmolality of the culture medium). Sodium hypophosphite did not show any clastogenic activity in the in vitro mammalian chromosome aberration test with cultured human lympocytes.

Executive summary:

The potential of sodium hypophosphite to induce chromosome aberrations in cultured human lymphocytes was assessed according to the OECD guideline 473 and the EU Method B.10. The study was conducted in compliance with the principles of Good Laboratory Practice on the monohydrated form of the test item as the anhydrous form is highly hygroscopic and difficult to handle without specific precautions.

Two experiments, with and without a metabolic activation system, were performed in duplicate and together with vehicle and positive controls. In the first experiment cultured human lympocytes were exposed to 19.53, 39.06, 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/mL of sodium hypophosphite for 3h and harvested 20 hours after the beginning of treatment. Cytotoxicity was recorded for all these concentrations, while only concentrations 625, 1250 and 2500 µg/mL were selected for the metaphase analysis. Since this first experiment gave negative results both with and without activation, a second experiment was performed as stated in the guidelines. In this second experiment, treatment in absence of S9 mix was continuous until harvest whilst cultured cells in the presence of S9 mix were exposed for 3 hours to sodium hypophosphite. Cells were always harvested 20 and 44 hours after the beginning of the treatment. The concentrations of test substance used in the second experiment were 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL to calculate the mitotic index and for metaphase analysis sodium hypophosphite concentrations of 625, 1250 and 2500 µg/mL was selected for the 20h treatments and 2500 µg/mL for the 44h treatments.

The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Sodium hypophosphite did not exhibit significant cytotoxicity and did not induce any statistically significant increases in the frequency of cells with aberrations in either of the two experiments for all tested concentrations (range: 19.53 - 2500 µg/ml, 2500 µg/mL was maximal practicable concentration regarding osmolality of the culture medium).

Under these test conditions sodium hypophosphite did not show any clastogenic activity in the in vitro mammalian chromosome aberration test with cultured human lympocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

GENETIC TOXICITY IN VITRO /SODIUM HYPOPHOSPHITE DATA:

1- Gene mutation in bacteria:

The mutagenic activity of sodium hypophosphite was assessed in the Ames test performed in five strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) according to the OECD guideline 471 and the EU Method B13/14. The study was in compliance with the Principles of Good Laboratory Practice. Sodium hypophosphite was tested in two independent experiments, with and without a metabolic activation system, both performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Bacteria were exposed to sodium hypophosphite at five dose-levels (three plates/dose-level) selected from a preliminary toxicity test. In the first experiment, the dose-levels ranged from 312.5 to 5000 µg/plate for all strains, with and without S9 mix. In the second experiment, the dose-levels ranged from 625 to 5000 µg/plate for all strains, with and without S9 mix. After 48 to 72 hours of incubation, the revertant colonies were scored.

The numbers of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered valid. Sodium hypophosphite did not induce any significant increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Sodium hypophosphite was considered to be non-mutagenic under the conditions of this test.

2- Chromosomal aberrations in mammalian cells:

The genotoxic activity of sodium hypophosphite was assessed in cultured human lymphocytes according to OECD guideline 473 and EU Method B.10. The study was in compliance with the Principles of Good Laboratory Practice. Sodium hypophosphite was tested in two independent experiments, with and without a metabolic activation system, together with vehicle and positive controls. In the first experiment, lymphocytes cultures were exposed to sodium hypophosphite or control items for 3 hours without or with S9 mix and cells were harvested 20 hours after the beginning of treatment. In the second experiment, treatment in absence of S9 mix was continuous until harvest whilst the cultured cells were exposed for 3 hours to sodium hypophosphite in the presence of S9 mix. Cells were harvested 20 and 44 hours after the beginning of the treatment.

The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Sodium hypophosphite did not exhibit significant cytotoxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range from 19.53 to 2500 µg/ml, the maximal practicable concentration regarding the osmolality of the culture medium.

Sodium hypophosphite was considered to be non-clastogenic to human lymphocytes in vitro.

3- Gene mutation in mammalian cells:

The mutagenic activity of sodium hypophosphite was assessed in L5178Y TK+/- mouse lymphoma cells according to the OECD guideline 476 and the EU Method B17. The study was in compliance with the Principles of Good Laboratory Practice. Sodium hypophosphite was tested in two independent experiments, with and without a metabolic activation system, together with vehicle and positive controls. As positive control methylmethanesulfonate was used in experiments without S9 mix and cyclophosphamide in the experiments with S9 mix. Cells as suspension cultures were exposed to sodium hypophosphite at six dose-levels (two cultures/dose-level) selected from a preliminary toxicity test. For both experiments the dose-levels ranged from 78.13 to 2500 µg/mL for all lymphoma cell cultures, with and without S9-mix. In the first experiment lymphoma cell cultures were exposed to sodium hypophosphite or control items for 3 hours without or with S9 mix. In the second experiment lymphoma cell cultures were exposed to sodium hypophosphite or control items for 3 hours in the presence of S9 mix and for 24 hours in the absence of S9 mix. In both experiments cells were harvested at the end of the treatment period and to enable the expression of the mutant genotype were incubated for 48 hours at 37°C in a humidified atmosphere. Afterwards cells were plated with trifluorothymidine as selection agent.

The cloning efficiency and mutation frequency of the vehicle controls and the increase in the mutation frequency of the positive controls were as specified in the acceptance criteria. The study was therefore considered valid. Sodium hypophosphite did not exhibit significant cytotoxicity and no noteworthy increase in the mutation frequency in either of the experiments, both without and with S9 mix, at any dose-level.

Sodium hypophosphite was considered to be non-mutagenic to mouse lymphoma cells in vitro.

Justification for classification or non-classification

According to the criteria laid down in EU regulation (EC) n° 1272/2008 (CLP) and the EU directive 67/548/EEC, Potassium phosphinate has not to be classified for genetic toxicity.