Ammonium phosphinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2020 to 22 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The study was performed to evaluate the mutagenic potential of test chemical using Salmonella typhimurium strains TA98, TA100, TA1537, TA1535 and TA102 in Trial I (with 10% v/v S9 mix and without metabolic activation) and Trial II (with 10% v/v S9 mix and without metabolic activation) experiments including the vehicle (distilled water) and concurrent positive controls in triplicates.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium phosphinate

EC Number:

232-266-0

EC Name:

Ammonium phosphinate

Cas Number:

7803-65-8

Molecular formula:

H3N.H3O2P

IUPAC Name:

ammonium phosphinate

Test material form:

solid

Remarks:

White hygroscopic crystals

Details on test material:

- Name of test material: Ammonium hypophosphite
- Molecular formula: H4NO2P
- Molecular weight: 83.0264 g/mol
- Subsatnce type: Inorganic
- Physical state: Solid
- Appearance: White hygroscopic crystals
- Purity: 97.2%

Method

Target gene:

Histidine Operon

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes (S9 homogenate) prepared in house were used for the assay
- method of preparation of S9 mix: S9 mix [Cofactors (Cofactor ingredients: D-glucose-6-phosphate, β-NADP, magnesium chloride, potassium chloride, sodium phosphate and Liver homogenate] was prepared prior to use in the study.
- concentration or volume of S9 mix and S9 in the final culture medium : 10 % (v/v) in the S9 mix.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

Test concentrations with justification for top dose:

0.3125, 0.625, 1.25, 2.5 and 5 mg/plate. Based on the result of cytotoxicity study, doses were selected.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water

- Justification for choice of solvent/vehicle: On the basis of solubility test, distilled water was selected as a vehicle for the study.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicate
- Number of independent experiments: The test chemical was tested in two independent experiments (Trial I and Trial II). Trial I: plate incorporation method and Trial II: preincubation method.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37 ± 2 °C
- Exposure duration/duration of treatment: 48 hours at 37 ± 2 °C
- Harvest time after the end of treatment (sampling/recovery times): No data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction of revertants per plate and/or clearing or diminution of the bacterial background lawn
- Any supplementary information relevant to cytotoxicity: No data

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- OTHER: No data

Rationale for test conditions:

No data available

Evaluation criteria:

A test item is considered as a mutagen if biologically relevant increase in the mean number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed (Table 4). A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant, if reproduced in an independent experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent experiment.

Statistics:

No data available

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: Soluble in water (upto 50 mg/ml)
- Precipitation and time of the determination: No precipitation was observed at the tested concentration of 5 mg/plate
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): On the basis of the solubility and precipitation test, the preliminary cytotoxicity test was performed at the concentrations of 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. In this assay, the tester strains TA98 and TA100 were exposed to the test item, the vehicle and the positive controls according to the plate incorporation method. Each concentration of the test item including the controls was tested in triplicates.
In tester strains TA98 and TA100, no reduction in revertant count and no inhibition in background lawn were observed at any of the concentrations, either in the presence or absence of metabolic activation, when compared to the vehicle control data (Table 1).

STUDY RESULTS
- Concurrent vehicle control data : vehicle control was within the acceptable range of historical data of lab.
- positive control data: The positive controls used in the study produced significant increases in the mean number of revertant colonies in all of the tester strains when compared to the control data, thus confirming the validity of the test system to detect specific mutagens in the presence and absence of a metabolic activation system.

Ames test:
- Signs of toxicity : please reffer the table attached in result section (Appendix 4.)
- Individual plate counts : please reffer the table attached in remark section (Appendix 1, Appendix 2, Appendix 3)
- Mean number of revertant colonies per plate and standard deviation (Table 1, Table 2, Table 3)

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table 1: Mean Revertant Colony Count – Preliminary Cytotoxicity Assay

Test Item Concentration (mg/plate)	TA 98				TA 100
	- S9		+ S9		- S9		+ S9
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	21.67 (NI)	2.31	21.33 (NI)	1.53	101.67 (NI)	7.51	99.33 (NI)	9.07
T1 0.0390625	20.67 (NI)	3.06	19.33 (NI)	3.21	96.33 (NI)	3.06	94.00 (NI)	3.00
T2 0.078125	19.67 (NI)	3.51	20.33 (NI)	2.31	102.67 (NI)	4.51	97.33 (NI)	6.66
T3 0.15625	21.33 (NI)	2.31	21.67 (NI)	2.08	96.00 (NI)	6.00	105.33 (NI)	10.02
T4 0.3125	20.33 (NI)	1.15	21.33 (NI)	2.08	94.33 (NI)	4.16	92.67 (NI)	4.51
T5 0.625	22.00 (NI)	2.65	19.00 (NI)	2.00	100.33 (NI)	5.51	95.33 (NI)	4.04
T6 1.25	21.00 (NI)	5.00	20.67 (NI)	2.08	97.67 (NI)	5.03	92.00 (NI)	4.36
T7 2.5	20.33 (NI)	1.53	20.33 (NI)	1.15	94.33 (NI)	4.51	97.33 (NI)	6.03
T8 5.0	20.33 (NI)	2.31	18.33 (NI)	2.08	97.67 (NI)	4.73	95.00 (NI)	7.21
PC	392.33	20.21	407.33	13.20	728.67	24.99	725.00	23.07

Key: VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, NI = No Inhibition.

Note: Since there was no reduction in revertant count and no background lawn inhibition at 5 mg/plate, Trial I (plate incorporation method) was performed with 5 mg/plate as a highest concentration.  

Positive control:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 (absence of metabolic activation)
Benzo[a]pyrene	:	TA98 and TA100 (presence of metabolic activation)

Table 2: Mean Revertant Colony Count Trial I

Plate Incorporation Method [Absence of metabolic activation (-S9)]
Test Item Concentration (mg/plate)	TA 1537		TA 1535		TA 102
	Mean	SD	Mean	SD	Mean	SD
VC	5.33	1.15	13.33	2.52	234.33	8.08
T1(0.3125)	6.33	0.58	12.67	2.89	219.33	3.51
T2(0.625)	4.00	1.00	13.67	1.15	219.67	12.22
T3(1.25)	5.67	2.31	14.33	0.58	234.67	7.09
T4(2.5)	5.33	1.15	13.00	2.00	223.00	4.00
T5(5.0)	5.67	1.15	14.33	1.15	233.00	12.29
PC	209.33	20.79	349.67	12.22	1431.67	88.01

Plate Incorporation Method [Presence of metabolic activation (+S9 10 % v/v S9 Mix)]
Test Item Concentration (mg/plate)	TA 1537		TA 1535		TA 102
	Mean	SD	Mean	SD	Mean	SD
VC	6.33	1.53	13.33	2.52	222.67	9.07
T1(0.3125)	6.00	0.00	14.33	1.15	229.33	8.74
T2(0.625)	7.33	1.53	13.67	1.15	224.00	5.57
T3(1.25)	7.00	1.00	13.67	0.58	232.00	6.24
T4(2.5)	6.00	0.00	14.67	2.31	223.00	14.73
T5(5.0)	6.33	0.58	13.67	1.15	212.00	6.24
PC	206.33	15.53	362.67	9.29	1474.00	38.74

Key: VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.

Positive Control:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine	:	TA1537 (absence of metabolic activation)
Mitomycin-C	:	TA102(absence of metabolic activation)
Benzo[a]pyrene	:	TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Table 3: Mean Revertant Colony Count Trial II

Preincubation Method [Absence of metabolic activation]
Test Item Concentration (mg/plate)	TA 1537		TA 1535		TA 102		TA 98		TA 100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	6.67	1.15	13.33	2.08	219.67	22.90	21.67	3.06	101.33	6.66
T1(0.3125)	5.67	1.15	13.33	0.58	220.33	25.40	21.33	2.08	99.00	9.17
T2(0.625)	6.67	1.15	11.67	1.15	217.67	17.90	20.67	3.21	104.00	8.19
T3(1.25)	6.33	1.53	13.33	1.53	225.67	15.95	21.33	2.08	94.00	3.61
T4(2.5)	6.33	0.58	13.67	1.15	225.33	10.02	20.33	2.00	97.33	8.50
T5(5.0)	7.00	1.73	12.33	0.58	222.67	9.07	19.67	3.79	99.67	4.93
PC	233.00	15.39	360.67	15.14	1586.00	9.02	266.33	9.17	732.00	14.00

Preincubation Method [Presence of metabolic activation (+S9 10% v/v S9 Mix)]
Test Item Concentration (mg/plate)	TA 1537		TA 1535		TA 102		TA 98		TA 100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	6.00	2.00	13.00	1.00	222.67	9.07	19.67	3.06	94.67	2.08
T1(0.3125)	7.33	1.15	12.33	1.53	222.67	13.87	22.33	2.08	97.33	8.50
T2(0.625)	5.33	0.58	13.67	1.15	223.00	4.00	20.67	3.21	99.67	4.93
T3(1.25)	7.33	0.58	16.33	0.58	225.33	8.62	20.67	2.08	97.00	8.72
T4(2.5)	6.67	1.15	13.67	1.15	222.00	9.85	22.00	2.00	105.00	9.85
T5(5.0)	6.67	0.58	12.00	1.73	219.67	3.06	21.33	3.79	95.33	9.45
PC	245.00	8.19	372.67	10.02	1593.33	71.67	426.00	9.17	794.67	18.93

Key: NC = Negative control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.

Positive Control:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine	:	TA1537 (absence of metabolic activation)
Mitomycin-C	:	TA102(absence of metabolic activation)
Benzo[a]pyrene	:	TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Table 4: Fold Increase

Trial I - Plate Incorporation Method
Test Item Concentration (mg/plate)	TA 1537		TA 1535		TA 102		TA 98		TA 100
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
VC	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
T1 (0.3125)	1.19	0.95	0.95	1.08	0.94	1.03	0.94	1.00	0.93	0.93
T2 (0.625)	0.75	1.16	1.03	1.03	0.94	1.01	1.02	0.89	0.99	0.96
T3 (1.25)	1.06	1.11	1.08	1.03	1.00	1.04	0.97	0.97	0.96	0.93
T4 (2.5)	1.00	0.95	0.98	1.10	0.95	1.00	0.94	0.95	0.93	0.98
T5 (5.0)	1.06	1.00	1.08	1.03	0.99	0.95	0.94	0.86	0.96	0.96
PC	39.25	32.58	26.23	27.20	6.11	6.62	18.11	19.09	7.17	7.30

Trial II – Preincubation Method
Test Item Concentration (mg/plate)	TA 1537		TA 1535		TA 102		TA 98		TA 100
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
VC	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
T1 (0.3125)	0.85	1.22	1.00	0.95	1.00	1.00	0.98	1.14	0.98	1.03
T2 (0.625)	1.00	0.89	0.88	1.05	0.99	1.00	0.95	1.05	1.03	1.05
T3 (1.25)	0.95	1.22	1.00	1.26	1.03	1.01	0.98	1.05	0.93	1.02
T4 (2.5)	0.95	1.11	1.03	1.05	1.03	1.00	0.94	1.12	0.96	1.11
T5 (5.0)	1.05	1.11	0.93	0.92	1.01	0.99	0.91	1.08	0.98	1.01
PC	34.95	40.83	27.05	28.67	7.22	7.16	12.29	21.66	7.22	8.39

Key: NC = Negative control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation.

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames test of test chemical, was conducted by the plate incorporation and preincubation methods The study was performed as per the OECD guideline No. 471 (Adopted: July 21, 1997 The study was performed to evaluate the mutagenic potential of test chemical using Salmonella typhimurium strains TA98, TA100, TA1537, TA1535 and TA102 in Trial I (with 10% v/v S9 mix and without metabolic activation) and Trial II (with 10% v/v S9 mix and without metabolic activation) experiments including the vehicle (distilled water) and concurrent positive controls in triplicates. Preliminary cytotoxicity assay was performed using the strains TA98 and TA100 with the concentrations 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate along with the vehicle and positive controls both in the presence and absence of metabolic activation in triplicates Based on the solubility test, distilled water was selected as a vehicle for the test item in the study.

In tester strains TA98 and TA100, no reduction in revertant count and no inhibition in background lawn were observed at any of the concentrations, either in the presence or absence of metabolic activation, when compared to the vehicle control data.On the basis of preliminary cytotoxicity test results, main study was performed with the following concentrations: Trial I and II: 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate.

Trial I was performed both in the presence (10% v/v S9 mix) and absence of metabolic activation system along with the vehicle and the positive control with the test item concentration spacing factor of 2. There was no increase in the number of revertant colonies and no inhibition in background lawn up to the highest concentration of 5 mg/plate in the presence (10% v/v S9 mix) or absence of metabolic activation, when compared to the vehicle control data.

Trial II was conducted to confirm the negative results observed in Trial I. Trial II was conducted in the presence and in the absence of metabolic activation system with all the tester strains along with the vehicle and concurrent positive controls according to the preincubation method.

There was no increase in the number of revertant colonies and no background lawn inhibition up to the highest concentration of 5 mg/plate in the presence (10% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control.

The number of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) were in the range of historical data of the lab.The positive controls used in the study produced significant increase in the mean number of revertant colonies in all of the tester strains compared to the control data, thus confirming the validity of the test system to detect specific mutagens in the presence and in the absence of a metabolic activation system.

From the above data It can be concluded that the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.