Ammonium dihydrogenorthophosphate

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:

27 April 2001 -17 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February 1998.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997).

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium phosphate (DAP)

IUPAC Name:

Diammonium phosphate (DAP)

Constituent 2

Reference substance name:

Diammonium hydrogenorthophosphate

EC Number:

231-987-8

EC Name:

Diammonium hydrogenorthophosphate

Cas Number:

7783-28-0

Molecular formula:

H3N.1/2H3O4P

IUPAC Name:

diammonium hydrogen phosphate

Test material form:

other: particles

Details on test material:

- Name of test material (as cited in study report): Diammonium Phosphate (DAP)
- Substance type: brown discrete particles
- Physical state: solid

Method

Target gene:

Salmonella tester strain cultures: deep rough mutation (ifa) and the deletion in the uvrB gene.
Cultures of tester strains TA98 and TA100: presence of the pKM101 plasmid R-factor.
All WP2 uvrA cultures: deletion in the uvrA gene

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:

other: TA98 + TA1537: reverted from histidine dependence to histidine independence by frameshift mutagens. TA1535: reverted by mutagens that cause basepair substitutions. TA100: reverted by mutagens that cause both frameshift and basepair substitution mutations

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:

other: Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9-mix from male Sprague-Dawley rats

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 ug per plate.
Confirmatory mutagenicity assay: 50, 150,500, 1500 and 5000 ug per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water (CAS# 7732-18-5), obtained from Life Technologies, Inc.
- Justification for choice of solvent/vehicle: Based on compatibility with the target cells and workability of the test article in water.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene: 1.0 and 10 ug/plate

Remarks:

All tester stains with S9 activation mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without S9 activation mix: 1.0 ug/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535 without S9 activation mix: 1.0 ug/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 activation mix: 75 ug/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without S9 activation mix: 1000 ug/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in top agar (plate incorporation)

DURATION
- Preincubation period: overnight (To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored)
- Exposure duration: approximately 48 to 72 hours at 37±2°C

NUMBER OF REPLICATIONS:
Initial Toxicity-Mutation Assay in duplicate
Confirmatory Mutagenicity Assay in triplicate

DETERMINATION OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.

OTHER EXAMINATIONS:
- Other: Precipitate was evaluated by visual examination without magnification.

OTHER:
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

Evaluation criteria:

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for tester strains TA98, TA1OO and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.
Since no positive responses were observed, no statistical analysis of the responses was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium 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:

not applicable

Positive controls validity:

valid

Test results 5

Key result

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:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitation conc: Initial Toxicity Mutation Assay: 1,800 μg/plate
Confirmatory Mutagenicity Assay: 5,000 µg/plate

Any other information on results incl. tables

Initial Toxicity-Mutation Assay

With (+) or Without (-) S9-mix	Test substance concentration (μg/plate)	Number of colonies/plate^a)
		Base-pair substitution type			Frame shift type
		TA100	TA1535	WP2uvrA		TA98	TA1537
-S9 mix	Solvent control	130	18	13		12	7
	2.5	101	12	12		10	4
	7.5	118	16	14		14	4
	25	103	17	11		19	6
	75	102	16	16		14	5
	200	128	14	11		13	7
	600	91	14	10		16	6
	1800	100 *	14 *	12 *		12 *	5 *
	5000	83 *	16 *	10 *		12 *	5 *
+S9 mix	Solvent control	145	21	14		15	9
	2.5	119	9	18		16	9
	7.5	124	11	14		18	6
	25	117	12	17		19	7
	75	99	11	14		13	6
	200	110	14	13		21	6
	600	92	12	17		15	7
	1800	90 *	13 *	19 *		17 *	9 *
	5000	95 *	15 *	9 *		13 *	6 *
Positive control without S9 mix	Name	NaN3	NaN3	MMS		2-NF	9-AA
	Number of colonies/plate	396	176	94		97	343
Positive control with S9 mix	Name	2-AA	2-AA	2-AA		2-AA	2-AA
	Number of colonies/plate	486	87	167		459	70

a) The average number of revertant colonies from two plates

*)Precipitate

NaN₃= sodium azide; 2-NF= 2-nitrofluorene; 9-AA = 9-aminoacridine; MMS = methyl methanesulfonate; 2 -AA = 2 -aminoanthracene

Solvent control = water

Confirmatory Mutagenicity Assay

With (+) or Without (-) S9-mix	Test substance concentration (μg/plate)	Number of colonies/plate^a)
		Base-pair substitution type			Frame shift type
		TA100	TA1535	WP2uvrA		TA98	TA1537
-S9 mix	Solvent control	119	20	16		12	6
	50	101	17	12		12	5
	150	136	17	15		9	5
	500	105	17	15		10	9
	1500	121	20	13		11	7
	5000	125 *	19 *	10 *		11 *	5 *
+S9 mix	Solvent control	148	13	19		20	8
	50	144	15	17		17	7
	150	125	15	12		18	8
	500	117	17	13		20	8
	1500	128	14	16		16	7
	5000	117 *	15 *	10 *		21 *	5 *
Positive control without S9 mix	Name	NaN3	NaN3	MMS		2-NF	9-AA
	Number of colonies/plate	475	244	100		113	475
Positive control with S9 mix	Name	2-AA	2-AA	2-AA		2-AA	2-AA
	Number of colonies/plate	1094	143	239		977	97

a) The average number of revertant colonies from three plates

*)Precipitate

NaN₃= sodium azide; 2-NF= 2-nitrofluorene; 9-AA = 9-aminoacridine; MMS = methyl methanesulfonate; 2 -AA = 2 -aminoanthracene

Solvent control = water

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Diammonium Phosphate (DAP) did not cause a positive response in the presence and absence of Aroclor-induced rat liver S9.