Polyphosphoric acids, ammonium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 19 March 2010 to 11 May 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conclusive, performed to a valid guideline (OECD TG 476, adopted 21 July 1997) and was conducted under GLP conditions. No deviations from the test methods were noted. The reliability has been assigned in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006) as part of a weight of evidence approach. Read-across in accordance with Annex XI, Section 1.5 of Regulation (EC) No. 1907/2006 (REACH) is justified on the following basis: Polyphosphoric acids, ammonium salt (also known as ammonium polyphosphate) is a mixture of oligomeric species of ammonium phosphate. When analysed the substance appears to consist mainly of ammonium orthophosphates, ammonium diphosphate and ammonium triphosphate. Phosphate is not considered to be mutagenic as it is frequently present within the buffers used for in vitro mutagenicity tests (i.e. as sodium or potassium phosphates). Therefore in order to assess the mutagenicity potential of ammonium polyphosphate it is the ammonium ion that is of interest. As such it is scientifically justified to read-across to other ammonium phosphates. Such as, ammonium dihydrogenorthophosphate and diammonium hydrogenorthophosphate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Polyphosphoric acids, ammonium salts

EC Number:

269-789-9

EC Name:

Polyphosphoric acids, ammonium salts

Cas Number:

68333-79-9

Molecular formula:

[NH4PO3]n

IUPAC Name:

undecaammonium bis(phosphonatooxy)phosphinate dihydrogen phosphate hydrogen (phosphonatooxy)phosphonate hydrogen phosphate

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): ammonium dihydrogenorthophosphate
- CAS Number: 7722-76-1
- Physical state: white crystals
- Analytical purity: 99.8%
- Lot/batch No.: 0927299
- Expiration date of the lot/batch: 03 February 2011 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: at room temperature in the dark
- Stability under storage conditions: stable

Method

Target gene:

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

Species / strain

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

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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.