Diammonium dihydrogenpyrophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key studies are avaialble on analogous substances. All studies are performed in accordance with an appropriate guideline and under the conditions of GLP.

Link to relevant study records

Reference

Reference 1

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:

From 12 May 1995 to 13 June 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The proposed source chemical (is a mixture of ammonium orthophosphates and ammonium pyrophosphates and is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates and pyrophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate or pyrophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus, the target substance (diammonium dihydrogenpyrophosphate) and the source substance (mixture of ammonium orthophosphates and pyrophosphates) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the toxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 31 January 1994 Date of signature on certificate: 16 March 1994

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The identity of the test material is not reported within the study report itself, however the data is referred to in the Toxicological Risks of Selected Flame Retardant Chemicals (2000), Subcommittee on Flame-Retardant Chemicals, Committee on Toxicology, Board on Environmental Studies and Toxicology, National Research Council. ISBN: 0-309-59232-1. The substance LR-2 is an ‘ammonium polyphosphate’ and the author provides the following additional information with regards to the chemical identity of LR2: ‘Based on information provided by the manufacturer (Stewart Miller, Albright and Wilson, pers. commun., Nov. 1, 1999), a typical species distribution of polyphosphates in LR2 is 20% orthophosphate, 40% pyrophosphate,

Target gene:

Histidine for Salmonella.

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA100, TA1537, TA1538, TA98

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0, 25, 75, 250, 750, 2500, 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 4-nitro-o-phenylenediamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Exposure duration: approximately 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of number of revertant colonies and examined for a thinning of the background lawn

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained, then a third experiment may be used to confirm the correct response. All data are statistically analysed using the methods recommended by UKEMS (5) and normally Dunnett's method of linear regression is used to evaluate the result. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA1538, TA98, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Study

The dose range of the test material used in the preliminary toxicity study was, (after a purity allowance), 0, 25, 75, 250, 750 and 2500 µg/plate. The maximum dose level was not achieved in the preliminary toxicity test, this is not considered significant as there was no toxicity noted in the main experiments at any dose level tested. The test material was non-toxic in the strain of Salmonella used (TA 100).

The mean numbers of revertant colonies for the toxicity assay were:

Strain	Dose (µg/plate)
	0	25	75	250	750	2500
TA100	110	107	100	123	117	109

Mutation Study

The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were aU found to be satisfactory.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls both with and without metabolic activation are presented in Tables 2 and 3 for experiment 1 and Tables 4 and 5 for experiment 2.

Key to Table of Test Results

1 . "Number of revertants" - The observed values and average value (in parentheses) are shown in order, beginning with the lowest concentration of the test substance.

2. Figures immediately below average values refer to standard deviation.

3 . The following postfixes are used when required:

C = contaminated

P = precipitate

S =sparse background lawn

v = very thin background lawn

T = toxic

x = plate unscorable

Table 1: Spontaneous Mutation Rates

Experiment 1:

Number of Revertants (Number of Colonies per plate)
Base-pair Substitution Type				Frameshift Type
TA100		TA1535		TA1538		TA98		TA1537
112		20		23		19		20
100	(98)	27	(23)	24	(22)	14	(17)	19	(17)
82		23		20		18		12

 

Experiment 2:

Number of Revertants (Number of Colonies per plate)
Base-pair Substitution Type				Frameshift Type
TA100		TA1535		TA1538		TA98		TA1537
116		22		15		22		12
105	(102)	17	(19)	13	(22)	27	(17)	12	(10)
85		19		20		17		5

Table 2:

Experiment 1 – Without Metabolic Activation

With or without S9-mix	Test substance concentration µg/plate	Number of revertants (Number of colonies per plate)
		Base-pair substitution type				Frameshift type
		TA 100		TA 1535		TA 1538		TA 98		TA 1537
-	0	153		25		15		22		17
		164	(149)	17	(22)	17	(18)	24	(21)	12	(12)
		131	16.8	24	4.4	23	4.2	18	3.1	8	4.5
-	25	144		17		19		17		9
		139	(137)	15	(16)	20	(16)	25	(23)	12	(8)
		129	7.6	15	1.2	10	5.5	28	5.7	4	4.0
-	75	136		13		23		23		14
		122	(125)	18	(14)	19	(18)	19	(21)	12	(12)
		118	9.5	12	3.2	12	5.6	22	2.1	9	2.5
-	250	137		17		27		27		9
		123	(127)	10	(15)	25	(19)	25	(23)	12	(9)
		121	8.7	18	4.4	17	1.0	17	5.3	5	3.5
-	750	125		19		14		14		9
		118	(132)	17	(19)	19	(18)	19	(18)	12	(10)
		154	19.1	20	1.5	20	4.0	20	3.2	8	2.5
-	2500	136		13		32		32		8
		127	(128)	15	(14)	27	(19)	27	(26)	10	(10)
		120	8.0	15	1.2	19	4.0	19	6.6	13	2.5
-	5000	90		10		19		19		8
		85	(80)	10	(9)	15	(21)	15	(19)	3	(6)
		64	14.0	8	1.2	22	4.9	22	3.5	8	2.9
Positive controls	Name	ENNG		ENNG		4NOPD		4NOD		9AA
	Concentration (µg/plate)	3		5		5		0.2		80
S9-mix -	Number of colonies per plate	869		312		296		172		447
		802	(794)	334	(308)	249	(267)	184	(182)	495	(444)
		712	79.8	277	28.7	255	25.5	189	8.7	391	52.1

ENNG = N-ethyl-N’-nitro-N-nitrosguanidine 4NOPD = 4-nitro-o-phenylenediamine

4NQO = 4-nitroquinoline-1-oxide 9AA = 9-aminoacridine

Table 3:

Experiment 1 – With Metabolic Activation

With or without S9-mix	Test substance concentration µg/plate	Number of revertants (Number of colonies per plate)
		Base-pair substitution type				Frameshift type
		TA 100		TA 1535		TA 1538		TA 98		TA 1537
+	0	105		25		33		25		18
		87	(98)	14	(16)	45	(32)	33	(31)	25	(24)
		103	9.9	10	47.8	17	14.0	34	4.9	28	5.1
+	25	106		17		33		41		23
		97	(100)	10	(15)	30	(27)	34	(36)	20	(24)
		97	5.2	19	4.7	17	8.5	32	4.7	28	4.0
+	75	105		25		30		35		22
		115	(109)	17	(19)	22	(25)	18	(29)	23	(23)
		105	5.5	15	5.3	19	5.7	33	9.3	25	1.5
+	250	111		19		25		33		20
		96	(103)	13	(15)	27	(26)	28	(28)	20	(21)
		101	7.6	12	3.8	27	1.2	22	5.5	24	2.3
+	750	96		13		32		19		14
		111	(103)	24	(19)	24	(29)	32	(27)	18	(15)
		101	7.6	19	5.5	32	4.6	29	6.8	14	2.3
+	2500	107		14		27		19		17
		116	(111)	10	(12)	36	(30)	34	(31)	23	(18)
		111	4.5	12	2.0	27	5.2	40	10.8	15	4.2
+	5000	105		12		29		28		12
		102	(97)	8	(10)	32	(28)	20	(27)	12	(14)
		84	11.4	9	2.1	22	5.1	33	6.6	17	2.9
Positive controls	Name	2AA		2AA		2AA		2AA		2AA
	Concentration (µg/plate)	1		2		0.5		0.5		2
S9-mix +	Number of colonies per plate	732		110		294		276		219
		803	(857)	139	(129)	191	(252)	220	(241)	232	(229)
		1035	158.5	138	16.5	271	54.1	227	30.5	229	6.8

2AA = 2-aminoanthracene

For Table 4 and 5 - Experiment 2, please see attached document

Conclusions:

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

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA99 and TA100 were treated with the test material using the Ames plate incorporation method at six 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 was determined in a preliminary toxicity assay and was, (after a purity allowance of approximately 50%), 25 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh chemical formulations. The method used conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B 14 in Commission Directive 92/69/EEC.

The vehicle (sterile distilled water) control plates produced counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

The test material caused no visible reduction in the growth of the bacterial lawn at any of the dose levels in any of the strains of Salmonella tested. The test material was therefore, tested up to the maximum recommended dose of 5000 µg/plate.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of this test

Reference 2

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus, the target substance (diammonium dihydrogenpyrophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the toxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

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 Migrated to IUCLID6: 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 Migrated to IUCLID6: 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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:

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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 March 2010 to 11 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The proposed source chemical (ammonium dihydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus, the target substance (diammonium dihydrogenpyrophosphate) and the source substance (ammonium dihydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the toxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1, 3, 10, 33, 100, 333, 750, 1150 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (RPMI medium) treatment groups were used as the vehicle controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (RPMI medium) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Two mutagenicity experiments were performed. In the first experiment, L5178Y TK mouse lymphoma cells were treated with the test material at eight dose levels, in duplicate, together with vehicle and positive controls. The exposure groups used were as follows: 3 hour exposures both with and without metabolic activation (8% S9-mix).

In the second experiment, L5178Y TK mouse lymphoma cells were treated with the test material at eight dose levels, in duplicate, together with vehicle and positive controls. The exposure groups used were as follows: 3 hour exposure with metabolic activation (12% S9-mix), and 24 hours without metabolic activation.

For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (if possible) were subcultered every day in oder to maintain log phase growth. Two days after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

The dose range of test material was selected following the results of a preliminary toxicity test and was 1 to 1150 µg/mL. There was no evidence of any marked toxicity in the preliminary toxicity test up to and including the highest test substance concentration of 1150 µL.

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Cell culture:

Horse serum was inactivated by incubation at 56 °C for at least 30 minutes.

Basic medium: RPMI 1640 Herpes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1mM sodium pyruvate and 2mM L-glutamin.

Growth medium: Basic medium, supplemented with 10% (v/v) heat-activated horse serum (=R10 medium)

Exposure medium: For 3 hour exposure: Cells were exposed to the test substance in basic medium supplemented wuith 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium: selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL triflurothymidine (TFT)

Non-selective medium: non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

Environmental conditions:
All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80-100% (actual range 59-94%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0 °C (actual range 34.9 - 37.9 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.1 - 36.0 °C), humidity (with a maximum of 9%) and CO2 percentage (with a maximum of 1%) occured that were caused the opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Evaluation criteria:

Data evaluation and statistical procedures:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for biological relevance including a comparison of the results with the historical control data range.

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 (ref. 12).

A test substance is considered positive (mutagenic) in the mutation assay if it induced a MF of more than 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 reached a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:

Table 1 shows the cell counts of the cultures after 3 hours of treatment with various concentrations of ammonium dihydrogenorthophosphate and after 24 hours and 48 hours of subculture and the calculated suspension growth and the relative suspension growth.

Both in the absence and presence of S9 -mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 1150 µg/mL compared to the suspension growth of the solvent control.

Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of ammonium dihydrogenorthophosphate and after 24 hours of subculture and the calculated suspension growth and relative suspension growth.

In the absence of S9 -mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 1150 µg/mL compared to the suspension growth of the solvent control.

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.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell lines

Remarks:

Migrated from field 'Test system'.

First mutagenicity test:

Table 3 shows the percentages of cell survival and the mutation frequencies for various concentrations of ammonium dihydrogenorthophosphate.

No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

No significant increase in the mutation frequency at the TK locus was observed after treatment with ammonium dihydrogenorthophosphate either in the absence or presence of S9-mix. The numbers of small and large colonies in the ammonium dihydrogenorthophosphate treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test:

Table 4 shows the percentages of cell survival and the mutation frequencies for various concentrations of ammonium dihydrogenorthophosphate.

No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

No significant increase in the mutation frequency at the TK locus was observed after treatment with ammonium dihydrogenorthophosphate either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the ammonium dihydrogenorthophosphate treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Table 1 – Dose range finding test; Cytotoxicity of ammonium dihydrogenorthophosphate (3 hours treatment)

dose (µg/mL)	cell count after 3 hours of treatment (cells/mL x 10^5)	cell count after 24 hours of subculture (cells/mL x 10^5)	cell count after 48 hours of subculture (cells/mL x 10^5)	SG^1(x 10^5cells/mL)	RSG^2(%)
without metabolic activation
SC	5.3	3.8	7.5	96	100
33	5.6	4.0	7.7	110	114
100	5.5	3.6	7.7	98	102
333	5.2	3.6	7.8	94	98
750	5.3	3.6	7.8	96	99
1150	5.1	3.4	7.9	87	90
with metabolic activation
SC	5.5	3.9	8.2	113	100
33	5.7	3.8	9.1	126	112
100	5.7	3.8	8.4	116	103
333	5.5	4.1	8.2	118	105
750	5.4	4.0	7.9	109	97
1150	5.3	3.6	8.7	107	95

 

 

Table 2 – Dose range finding test; Cytotoxicity of ammonium dihydrogenorthophosphate (24 hours treatment)

dose (µg/mL)	cell count after 3 hours of treatment (cells/mL x 10^5)	cell count after 24 hours of subculture (cells/mL x 10^5)	SG^1(x 10^5cells/mL)	RSG^2(%)
without metabolic activation
SC	5.7	8.1	37	100
33	6.1	8.1	40	107
100	6.4	7.5	38	104
333	5.9	7.7	37	100
750	4.9	7.5	29	80
1150	4.9	6.5	25.69

 

Note: all calculations were made without rounding off

SC = solvent control = RPMI medium

(1)   

(2)   

 

Table 3 – Dose range finding test: Cytotoxic and mutagenic response of ammonium dihydrogenorthophosphate in the mouse lymphoma L5178Y test system

dose (µg/mL)	RSG (%)	CEday2(%)	RSday2(%)	RTG (%)	Mutation frequency x 10^-6
					total	small	large
without metabolic activation: 3 hours treatment
SC1	100	91	100	100	101	51	45
SC2		95			104	55	43
1	109	99	107	116	93	52	37
3	107	95	102	109	108	48	54
10	110	93	99	109	109	59	44
33	117	111	119	140	94	56	34
100	104	98	105	109	82	43	35
333	101	83	89	89	74	40	31
750	101	99	107	107	95	41	49
1150	93	102	110	102	84	47	34
MMS	67	54	58	39	1095	607	338
with 8% (v/v) metabolic activation: 3 hours treatment
SC1	100	97	100	100	106	54	46
SC2		97			83	38	41
1	112	105	109	122	82	45	34
3	112	93	96	107	94	41	49
10	99	99	103	102	88	55	29
33	99	94	97	96	85	49	33
100	100	101	104	104	90	51	35
333	95	98	101	96	94	58	32
750	88	97	100	88	97	53	39
1150	89	101	104	93	118	67	44
CP	73	48	49	36	1809	877	564

 

Table 4 – Experiment 2: Cytotoxic and mutagenic response of ammonium dihydrogenorthophosphate in the mouse lymphoma L5178Y test system

dose (µg/mL)	RSG (%)	CEday2(%)	RSday2(%)	RTG (%)	Mutation frequency x 10^-6
					total	small	large
without metabolic activation: 24 hours treatment
SC1	100	80	100	100	95	60	32
SC2		65			79	51	28
1	109	72	100	108	69	20	48
3	112	58	80	90	96	57	36
10	116	60	83	96	95	61	32
33	116	59	82	94	81	30	49
100	112	58	80	90	59	36	22
333	98	49	67	65	69	35	33
750	77	51	70	55	89	56	31
1150	58	45	61	35	134	60	70
MMS	73	37	51	37	1424	851	404
with 12% (v/v) metabolic activation: 3 hours treatment
SC1	100	65	100	100	88	53	33
SC2		72			89	46	40
1	94	58	85	80	120	74	42
3	95	120	174	165	38	21	17
10	91	60	88	79	78	37	39
33	97	58	84	81	110	66	41
100	88	48	69	61	160	110	44
333	90	97	140	127	85	54	28
750	90	60	88	79	115	65	46
1150	69	41	60	42	127	75	49
CP	15	33	48	7	935	668	212

 

Note: all calculations were made without rounding off

RSG = Relative suspension growth; CE = cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = RPMI 1640 medium; MMS = methylmethanesulfonate; CP = cyclophosphamide

Conclusions:

Ammonium dihydrogenorthophosphate is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The report describes the effects of ammonium dihydrogenorthophosphate on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and β-napthaflavone).

In the first experiment, ammonium dihydrogenorthophosphate was tested up to concentrations of 1150 µg/mL (0.01 M) in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. In the second experiment, ammonium dihydrogenorthophosphate was again tested up to concentrations of 1150 µg/mL, but in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. No toxicity was observed at this dose level in the absence and presence of S9-mix.

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

Mutation frequencies in cultures treated with positive control chemicals were increased 11-fold and 16-fold for MMS in the absenceof 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 theabsence 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substance has no structural alerts for mutagenicity. Furthermore, in vitro studies from analogous substances were all negative. These studies were performed in accordance with an approved guideline and under the conditions of GLP. All studies were negative both with and without metabolic activation. There is no reason to believe that these results would not be applicable to humans.

Justification for classification or non-classification

Based on the classification criteria and an assessment of the structure of diammonium dihydrogenpyrophosphate the substance is not considered to be mutagenic and no classification is proposed.