Phosphoric acid, potassium salt (2:3), dihydrate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tripotassium trihydrogen phosphate dihydrate

EC Number:

700-282-1

Cas Number:

66922-99-4

Molecular formula:

H304P.H20.3/2K

IUPAC Name:

tripotassium trihydrogen phosphate dihydrate

Details on test material:

- Name of test material (as cited in study report): NovaPK
- Physical state: white powder (Deseription from the Sponsor : Colourless crystalline powder)
- Lot/batch No.: 644
- Expiration date of the lot/batch: 23-Sep-2008
- Storage condition of test material: room temperature
- Other: container -opaque plastic bottle
- Analytical purity: no data

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9, induced with phenobarbitone/ß-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.
Main Study: 5000, 2500, 1250, 625 and 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
DMSO (for positive controls 9-aminoacridine, 2-nitrofluorene, 2-aminoanthracene)
Sterile distilled water (for the test item and the positive controls sodium azide and methylmethanesulphonate)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without activation

Migrated to IUCLID6: 1 µg/plate for TA100 and TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without activation

Migrated to IUCLID6: 50 µg/plate for TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without activation

Migrated to IUCLID6: 2 µg/plate for TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without activation

Migrated to IUCLID6: 500 µg/plate for WP2 uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene: 1 µg/plate for TA98 and TA100 (plate incorporation method), TA1535 and TA1537, 2 µg/plate for TA98 and TA100 (pre-incubation method), 10 µg/plate for WP2 uvrA (plate incorporation method, 20 µg/plate for WP2 uvrA (pre-incubation method)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, main test I); preincubation (main test II)

DURATION
- Preincubation period: 30 min
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 per concentration and experiment (main test)

DETERMINATION OF CYTOTOXICITY
- Method: decline in the number of spontaneous revertants, thinning of the background lawn or a microcolony formation.

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Regression analysis, Student's t-test

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no


RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed with any tester strain, in the absence or presence of S9 metabolic activation up to 5000 µg/plate.


COMPARISON WITH HISTORICAL CONTROL DATA:
mean plate counts for negative and positive controls fell within the normal historical range.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1, plate incorporation method, without metabolic activation

S9	Concentration (µg/plate)	Number of revertants (mean number of colonies / plate)
		TA1535	TA1537	TA98	TA100	WP2uvrA
-	0	18	16	27	138	29
-	313	17	15	26	151	25
-	625	21	15	25	140	29
-	1250	17	18	27	151	24
-	2500	17	22	28	147	22
-	5000	21	18	25	144	24
Negative control	Untreated	18	-	-	138	29
	DMSO (100µl/plate)	-	17	26	-	-
Positive controls	Name	SA	9-AA	2-NF	SA	MMS
	Concentration (µg/plate)	1	50	2	1	500
	No. of revertants (mean)	524	163	174	526	172

SA: Sodium azide

9-AA: 9-aminoacridine

MMS: methylmethane sulfonate

2-NF: 2-Nitrofluorene

 

Table 2: Experiment 1, plate incorporation method, with metabolic activation

S9	Concentration (µg/plate)	Number of revertants (mean number of colonies / plate)
		TA1535	TA1537	TA98	TA100	WP2uvrA
+	0	16	19	39	153	33
+	313	19	21	38	161	29
+	625	22	19	36	163	33
+	1250	21	20	38	169	27
+	2500	16	19	37	156	29
+	5000	17	19	36	159	28
Negative control	Untreated	-	-	-	-	-
	DMSO (100µl/plate)	20	19	33	151	30
Positive controls	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration (µg/plate)	1	1	1	1	10
	No. of revertants (mean)	118	100	469	1135	174

2-AA: 2-aminoanthracene

 

 

 

Table 3: Experiment 2, pre-incubation method, without metabolic activation

S9	Concentration (µg/plate)	Number of revertants (mean number of colonies / plate)
		TA1535	TA1537	TA98	TA100	WP2uvrA
-	0	18	16	28	152	28
-	313	18	16	29	152	28
-	625	16	16	31	155	28
-	1250	16	17	26	158	25
-	2500	19	18	27	154	27
-	5000	20	17	27	157	26
Negative control	Untreated	17	-	-	152	28
	DMSO (50µl/plate)	-	18	30	-	-
Positive controls	Name	SA	9-AA	2-NF	SA	MMS
	Concentration (µg/plate)	1	50	2	1	500
	No. of revertants (mean)	517	203	162	565	162

SA: Sodium azide

9-AA: 9-aminoacridine

MMS: methylmethane sulfonate

2-NF: 2-Nitrofluorene

 

Table 4: Experiment 2, pre-incubation method, with metabolic activation

S9	Concentration (µg/plate)	Number of revertants (mean number of colonies / plate)
		TA1535	TA1537	TA98	TA100	WP2uvrA
+	0	17	19	37	167	31
+	313	19	18	38	167	37
+	625	16	18	36	167	36
+	1250	16	18	40	167	38
+	2500	19	19	36	171	36
+	5000	18	20	38	160	36
Negative control	Untreated	-	-	-	-	-
	DMSO (50µl/plate)	16	19	38	126	33
Positive controls	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration (µg/plate)	1	1	1	1	10
	No. of revertants (mean)	113	106	479	1077	200

2-AA: 2-aminoanthracene

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation