Disodium fluorophosphate

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:

The experimental phase of this study was performed between 26 January 2010 and 10 February 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study is conducted according to an appropriate guideline and under the conditions of GLP, the study is therefore considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15 September 2009. Date of signature on GLP certificate: 26 November 2009.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium fluorophospahte

IUPAC Name:

Disodium fluorophospahte

Constituent 2

Reference substance name:

Disodium fluorophosphate

EC Number:

233-433-0

EC Name:

Disodium fluorophosphate

Cas Number:

10163-15-2

Molecular formula:

FH2O3P.2Na

IUPAC Name:

disodium fluorophosphate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor’s identification:Disodium fluorophosphate
Description : White solid
Batch number :9-01941-56
Date received :12 August 2009
Expiry date :January 2011
Storage conditions:Room temperature in the dark over silica gel

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment one (range-finding test): 50, 150, 500, 1500 and 5000 µg/plate
Experiment two (main test): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation
Dunnett's Linear Regression Analysis

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

Preliminary ToxicityTest

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA^-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	116	104	112	93	135	115	119	116	108	119	123
+	TA100	101	118	109	118	105	105	109	98	101	103	127
-	WP2uvrA^-	24	31	25	41	25	25	26	20	20	25	28
+	WP2uvrA^-	25	24	26	21	18	24	29	29	25	29	28

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented inTable1and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare and graphs are presented in Overall remarks, attachments.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1               Spontaneous Mutation Rates (Concurrent Negative Controls

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
100		20		21		22		5
102	(101)	18	(19)	16	(20)	22	(22)	7	(7)
100		18		24		21		9

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
79		9		19		24		11
98	(93)	12	(11)	23	(20)	10	(18)	9	(11)
102		12		18		19		12

 

Table2               Test Results: Range-Finding Test– Without Metabolic Activation

 

Test Period		From: 02 February 2010					To: 05 February 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
-	0	123 89 110	(107) 17.2#	15 24 24	(21) 5.2	25 24 24		(24) 0.6	19 18 15	(17) 2.1	9 11 15	(12) 3.1
-	50	104 86 97	(96) 9.1	21 25 17	(21) 4.0	20 25 25		(23) 2.9	20 16 15	(17) 2.6	12 8 11	(10) 2.1
-	150	100 104 115	(106) 7.8	19 25 18	(21) 3.8	24 25 17		(22) 4.4	16 24 18	(19) 4.2	11 11 11	(11) 0.0
-	500	86 95 105	(95) 9.5	24 22 24	(23) 1.2	22 22 19		(21) 1.7	18 17 24	(20) 3.8	13 6 9	(9) 3.5
-	1500	95 98 92	(95) 3.0	22 24 24	(23) 1.2	25 25 25		(25) 0.0	18 24 20	(21) 3.1	7 15 15	(12) 4.6
-	5000	108 111 83	(101) 15.4	20 26 27	(24) 3.8	18 18 19		(18) 0.6	21 20 22	(21) 1.0	11 13 10	(11) 1.5
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		552 593 439	(528) 79.8	436 460 470	(455) 17.5	645 681 685		(670) 22.0	152 154 149	(152) 2.5	735 691 703	(710) 22.7

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Table3               Test Results: Range-Finding Test– With Metabolic Activation

Test Period		From: 02 February 2010					To: 05 February 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
+	0	96 82 83	(87) 7.8#	18 14 15	(16) 2.1	19 25 19		(21) 3.5	24 25 25	(25) 0.6	11 10 15	(12) 2.6
+	50	83 84 88	(85) 2.6	19 14 16	(16) 2.5	21 18 20		(20) 1.5	22 24 27	(24) 2.5	10 10 11	(10) 0.6
+	150	95 95 78	(89) 9.8	12 15 13	(13) 1.5	25 15 27		(22) 6.4	17 26 27	(23) 5.5	9 11 10	(10) 1.0
+	500	86 85 85	(85) 0.6	17 18 14	(16) 2.1	18 17 18		(18) 0.6	16 24 21	(20) 4.0	7 14 11	(11) 3.5
+	1500	97 90 83	(90) 7.0	14 15 15	(15) 0.6	20 19 19		(19) 0.6	18 18 17	(18) 0.6	6 14 7	(9) 4.4
+	5000	91 93 90	(91) 1.5	15 19 15	(16) 2.3	26 26 28		(27) 1.2	25 21 22	(23) 2.1	8 17 18	(14) 5.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1188 1298 1294	(1260) 62.4	252 225 227	(235) 15.0	421 440 391		(417) 24.7	194 188 138	(173) 30.7	244 214 222	(227) 15.5

BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

#        Standard deviation

Table4               Test Results: Main Test– Without Metabolic Activation

 

Test period			From: 07 February 2010						To: 10 February 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
-	0	91 87 98	(92) 5.6#	13 21 23	(19) 5.3	29 18 19		(22) 6.1	19 22 18	(20) 2.1	10 20 13	(14) 5.1
-	50	107 100 98	(102) 4.7	16 21 31	(23) 7.6	24 14 26		(21) 6.4	12 23 20	(18) 5.7	19 12 23	(18) 5.6
-	150	100 91 90	(94) 5.5	18 19 22	(20) 2.1	16 22 21		(20) 3.2	20 23 34	(26) 7.4	12 18 16	(15) 3.1
-	500	66 88 68	(74) 12.2	15 8 11	(11) 3.5	23 27 14		(21) 6.7	16 18 15	(16) 1.5	12 11 12	(12) 0.6
-	1500	70 88 91	(83) 11.4	15 14 18	(16) 2.1	18 16 29		(21) 7.0	15 14 14	(14) 0.6	10 16 14	(13) 3.1
-	5000	100 87 78	(88) 11.1	11 14 18	(14) 3.5	18 27 19		(21) 4.9	12 11 11	(11) 0.6	11 11 7	(10) 2.3
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		405 434 446	(428) 21.1	1740 1797 1525	(1687) 143.4	468 558 475		(500) 50.1	106 84 109	(100) 13.7	538 674 665	(626) 76.1

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Table5               Test Results: Main Test– With Metabolic Activation

Test period		From: 07 February 2010					To: 10 February 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
+	0	77 87 109	(91) 16.4#	16 11 18	(15) 3.6	27 20 35		(27) 7.5	16 16 27	(20) 6.4	9 20 14	(14) 5.5
+	50	77 87 109	(91) 16.4	10 14 23	(16) 6.7	19 19 31		(23) 6.9	30 19 18	(22) 6.7	12 12 20	(15) 4.6
+	150	85 91 103	(93) 9.2	16 9 9	(11) 4.0	24 23 23		(23) 0.6	24 24 24	(24) 0.0	18 7 26	(17) 9.5
+	500	82 85 97	(88) 7.9	13 14 5	(11) 4.9	19 14 27		(20) 6.6	18 24 23	(22) 3.2	11 15 13	(13) 2.0
+	1500	73 78 151	(101) 43.7	7 8 8	(8) 0.6	22 30 25		(26) 4.0	31 19 20	(23) 6.7	15 14 13	(14) 1.0
+	5000	120 59 85	(88) 30.6	9 5 8	(7) 2.1	16 23 16		(18) 4.0	33 25 16	(25) 8.5	11 9 10	(10) 1.0
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1000 896 687	(861) 159.4	245 228 217	(230) 14.1	213 211 211		(212) 1.2	198 293 254	(248) 47.8	220 198 179	(199) 20.5

BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

#        Standard deviation

PLEASE SEE ATTACHED IN OVERALL REMARKS, ATTACHMENTS 1) Figures1-4 Dose-Response Curves. 2) Pages 23 -30 (Appendix 1 Report of Results in Mutagenicity Test using Micro-organisms).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

This study is conducted according to an appropriate guideline and under the conditions of GLP, the study is therefore considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.

Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrA^-were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.