Sodium phosphinate

Genetic toxicity: in vitro

Administrative data

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

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

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 in the form of monohydrate as the anhydrous form (CAS 7681-53-0) is highly hygroscopic and difficult to handle without specific precautions.
- Physical state: white crystalline solid
- Supplier: Rhodia UK-Oldbury
- Batch number: 90113D
- Expiry date: June 2010
- Storage condition of test material: room temperature, stored in a tightly closed container, opening of the container restricted to the minimum needs
- 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

Method

Species / strain

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

Controls

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

Results and discussion

Test results

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'.

Any other information on results incl. tables

- 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

Applicant's summary and conclusion

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.