Ammonium dihydrogenorthophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

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

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

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 / 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 / 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.

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

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

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

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

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.

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 3, 10, 33, 100 and 333 µg/ml and 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 3 µg/mL
Experiment 1:
Without S9-mix, 3 hours treatment: 0.003, 0.03, 0.1, 0.25, 0.5, 1, 1.4 and 2 µg/mL
With S9-mix, 3 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 12 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.01, 0.03, 0.1, 0.25, 0.5, 1, 1.4 and 1.8 µg/mL
With S9-mix, 3 hours treatment: 0.01, 0.1, 1, 10, 12, 14, 16 and 17 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium
- Justification for choice of solvent/vehicle:Test compound was soluble in RPMI 1640 medium and RPMI 1640 medium has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Positive control substance:

cyclophosphamide

Remarks:

with S9: 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

TEST SYSTEM
-cells: L5178Y/TK+/-3.7.2C

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplo cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more then MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 850 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 3 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 33 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 1 µg/mL in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 79 and 83% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level in both experiments.

Conclusions:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 16-fold for MMS in the absence of S9-mix, and by 19- and 11-fold for CP in the presence of
S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that Ammonium dihydrogenorthophosphate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 May 2001 - 12 June 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well-documented, GLP-compliant, according to OECD 473 in Chinese hamster ovary cells.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(Ninth Addendum; February 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 units penicillin and 100 ug streptomycin/mL and 2 mM L-glutamine for the non-activated study. S9 reaction mixture (3.5 mL serum-free McCoy's 5A medium (supplemented with 100 units penicillin and 100 ug streptomycin/mL and 2 mM L-glutamine) plus 1 mL of S9/cofactor pool) for the activated study. S9/cofactor pool = 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 uL S9 per milliliter serum-free McCoy's 5A medium supplemented with 100 units penicillin and 100 ug streptomycin/mL, and 2 mM L-glutamine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9: from male Sprague-Dawley rats induced with a single ip injection of Aroclor 1254, 500 mglkg, five days prior to sacrifice. S9 was batch prepared and stored frozen at approximately -70C until used.

Test concentrations with justification for top dose:

165, 330, 660 and 1320 μg/mL (2 replicates per concentration)

Vehicle / solvent:

Water
- Vehicle(s)/solvent(s) used: sterile water (CAS No.: 7732-18-5) obtained from Life Technologies Company.
- Justification for choice of solvent/vehicle: A solubility test was conducted to select the solvent. The test was conducted using purified water. The test article was tested to determine the solvent, selected in order of preference, that permitted preparation of the highest soluble stock concentration, up to 50 mg/mL for aqueous vehicles.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile water at the same concentrations as that found in the test article-treated groups

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

non-activated test system: 0.1 and 0.2 ug/ml final concentrations

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile water at the same concentrations as that found in the test article-treated groups

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

S9 activated test system: 10 and 20 ug/ml final concentrations

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16-24 hours.
- Exposure duration: In the non-activated study, the cells were exposed to the test article for 4 hours or continuously for 20 hour. In the S9 activated study, the cells were exposed for 4 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours



STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2 replicates per test substance concentration

NUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) were examined

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: viability of cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Mitotic index = number mitotic figures x 100/500 cells counted.
%Aberrant Cells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps.
Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures include quadriradials, triradials and complex rearrangements.
Chromosome breaks include breaks and acentric fragments; Die, dicentric chromosome.
Severely damaged cells includes cells with one or more pulverized chromosome and cells with 10 or more aberrations.

Evaluation criteria:

The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control. The number and types of aberration
found, the percentage ofstructurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated.
All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a
positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations
being statistically significant (p≤0.05). However, values that are statistically significant but do not exceed the range of historic solvent controls may
be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations will be
concluded to be negative.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent
aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level,
the Cochran-Armitage test was used to measure dose-responsiveness.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see Additional information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test article in treatment medium was
approximately 7.5
- Effects of osmolality: treatment medium of the highest concentration tested, 1320 ug/mL, was 292 mmol/kg. The osmolality in treatment of the highest soluble dose, 660 ug/mL, was 268 mmol/kg. The osmolality of the solvent (water) in treatment medium was 276 mmol/kg.
- Water solubility: The test article was soluble in treatment medium at dose levels ≤660 ug/mL.
- Precipitation: Visible precipitate was observed in treatment medium at dose level 1320 ug/mL.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 0.132 ug/mL to 1320 ug/mL in the absence and
presence of an S9 reaction mixture. The test article was soluble in treatment medium at dose levels 996 ug/mL. Visible precipitate was observed in treatment medium at dose level 1320 ug/mL.
Cell growth inhibition relative to the solvent control was 25% and 17% at 1320 ug/mL, the highest concentration tested in both the non-activated
and S9 activated 4 hour exposure groups, respectively. Cell growth inhibition relative to the solvent control was 37% at 1320 ug/mL, the highest
concentration tested in the non-activated 20 hour continuous exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA:
Both structural and numerical abberations of the cultures treated with the test articel were within ranges of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxicity of Diammonium Phosphate (DAP) (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the
absence of S9 activation was 56% at 660 ug/mL, the highest test concentration evaluated for chromosome aberrations.
Toxicity of Diammonium Phosphate (DAP) (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the
presence ofS9 activation was 5% at 1320 ug/mL, the highest test concentration evaluated for chromosome aberrations.
Toxicity of Diammonium Phosphate (DAP) (cell growth inhibition relative to the solvent control) in CHO cells when treated for 20 hours in the
absence of S9 activation was 63% at 660 ug/mL, the highest test concentration evaluated for chromosome aberrations.

Treatment	S9 activation	Treatment time	Mean mitotic index	Cells scored	Aberrations per cell (Mean +/- SD)	Cells with Aberrations
						Numerical (%)	Structural (%)
Water	-	4	16.0	200	0.000±0.000	0.5	0.0
DAP
165 µg/ml	-	4	14.1	200	0.005±0.071	0.0	0.5
330 µg/ml	-	4	11.6	200	0.005±0.071	2.0	0.5
660 µg/ml	-	4	6.7	200	0.015±0.158	3.0	1.0
MMC 0.2 µg/ml	-	4	15.0	200	0.105±0.380	0.5	8.5**
Water	+	4	17.1	200	0.025±0.186	1.5	2.0
DAP
330 µg/ml	+	4	15.9	200	0.005±0.071	2.5	0.5
660µg/ml	+	4	15.6	200	0.045±0.231	2.5	4.0
1320 µg/ml	+	4	16.4	200	0.015±0.122	1.5	1.5
CP 10 µg/ml	+	4	14.1	100	0.770±0.962	1.5	48.0**
Water	-	20	11.8	200	0.000±0.000	1.5	0.0
DAP
165 µg/ml	-	20	10.1	200	0.005±0.071	1.5	0.5
330 µg/ml	-	20	7.3	200	0.025±0.157	4.5	2.5*
660 µg/ml	-	20	6.3	200	0.005±0.071	1.0	0.5
MMC 0.1 µg/ml	-	20	11.4	200	0.200±0.521	0.5	15.5**

Treatment: Cells from all treatment conditions were harvested at 20 hours after the initiation of the treatments.

Aberrations per cell: Severely damaged cells were counted as 10 aberrations.

Percent aberrant cells: *, p≤0.05; **, p≤0.01; using Fisher's exact test

DAP = Diammonium Phosphate; CP = cyclophosphamide; MMC = mitomycin C

Conclusions:

Interpretation of results: negative

The positive and solvent controls fulfilled the requirements for a valid test.
Under the conditions of the assay described in this report, Diammonimn Phosphate (DAP) was concluded to be negative for the induction of
structural and numerical chromosome aberrations in CHO cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

With ammonium dihydrogenorthophosphate no in vitro studies were present for the Ames and the chromosome aberration tests, however with diammonium hydrogenorthophosphate studies are present. A reliable TK-assay with this substance itself was present.

In an Ames test according to OECD 471 guideline, Salmonella Typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E. Coli WP2 uvr A showed no genotoxicity with and without metabolic activation, tested up to the limit concentration. In an in vitro chromosome aberration test with CHO cells performed according to OECD 473 guideline, also no genotoxicity was seen with and without metabolic activation, while cytotoxicity was present. With ammonium dihydrogenorthophosphate a reliable TK-assay was present. In a Thymidine kinase (TK) assay in L5178Y mouse lymphoma cells performed according to OECD 476 and EC B.17 guidelines, the substance did not induce a significant increase in the mutation frequency. Based on these negative results for genotoxicity in in vitro studies, no in vivo studies are necessary.

Justification for selection of genetic toxicity endpoint
An Ames test and a chromosome aberration study with the read-across substance diammonium hydrogenorthophosphate are available, showing no adverse effects. A reliable Mouse lymphoma study with the substance is available, showing no adverse effects.

Short description of key information:
No reliable Ames and chromosome aberration studies with ammonium dihydrogenorthophoshate are present. Based on reliable in vitro studies with diammonium hydrogenorthophosphate, the Ames test and the chromosome aberration study were negative in the presence and absence of metabolic activation. For the in vitro TK assay a reliable study with ammonium dihydrogenorthophosphate is present showing negative results in the presence and absence of metabolic activation. The read-across rationale can be found in the category approach document attached in IUCLID Section 13 and fully included in the CSR.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, ammonium dihydrogenorthophosphate does not have to be classified according to regulation (EC) No.1272/2008 for genetic toxicity.