Tetrapotassium pyrophosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 September 2016 - 07 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

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

(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/phosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO34- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO34- anion. In biological systems pyrophosphates will be metabolised by intestinal alkaline phosphatase and it is assumed that the majority of diphosphate is absorbed as orthophosphate and thus both the target and source substances contain the same breakdown products.
(3) Potassium and sodium cations and phosphate anions are essential micronutrients and as such, their uptake is tightly regulated and is therefore not considered to pose a risk for genotoxicity.

It is therefore deemed scientifically justified to avoid any further testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted September 26, 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 16-19 April 2013 Date on Certificate: 14 May 2014

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

7758-80-7

Cas Number:

7758-80-7

IUPAC Name:

7758-80-7

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1531481
- Expiration date of the lot/batch: 19/07/2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in a tightly closed container and stored in a cool, dry and well-ventilated place.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test item was completely dissolved in highly purified water


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Fresh preparations of the test item were prepared on the day of the experiment and used for the treatment in all experimental parts.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral blood
- Suitability of cells: cells identified in OECD guideline.
- Sex, age and number of blood donors if applicable: Healthy non-smoking male of female individuals (18-35 years). No known recent exposures to genotoxic chemicals or radiation.
- Whether whole blood or separated lymphocytes were used if applicable:


Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B
The concentration used for this assay was 5 μg/mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

In the preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Sodium dihydrogen orthophosphate/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium.

Hence, 2000 µg/mL medium were employed as top concentration in the main study for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure)

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4hours and 24 hours at +37°C
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: 2 (main study), 1 (preliminary test)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide and left to air-dry.

NUMBER OF CELLS EVALUATED: The micronucleus frequencies were analysed in at least 2000 binucleate cells per concentration


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: relative total growth (relative increase in cell counts, RI); cytotoxicity = 100% - RI [%]

Evaluation criteria:

If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei, it is classified as a positive result.
Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolarity : No relevant changes in pH or osmolality of the test item formulations at concentrations of 3.16 to 2000 µg/mL medium were noted
- Water solubility: Soluble in water

RANGE-FINDING/SCREENING STUDIES: . In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Sodium dihydrogen orthophosphate/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium. Hence, 2000 µg/mL were employed as the top concentration for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Cytokinesis block proliferation index ranged between 1.26-1.48 for the test substance

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see tables below


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (micronucleus frequencies per 1000 cells):
Mitomycin C:
Mean: 46.9
SD: 35
range 17-137
Colchicine:
Mean: 25.9
SD: 9.5
range 15-63
cyclophosphamide:
Mean: 44.0
SD: 37.7
range 14-158
- Negative (solvent/vehicle) historical control data:

Without metabolic activation
Untreated control
mean 6.6
SD 2.9
range 2.0 - 17
95% Confidence interval 5.9 - 7.3

Vehicle control
mean 6.3
SD 3.1
range 2.0 - 18
95% Confidence interval 5.7 - 6.8

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI or RI in the case of the cytokinesis-block method

Any other information on results incl. tables

Table 1: Experiments without metabolic activation (S9 mix)

4 hr exposure
Concentration [µg/mL medium]	CBPI	RI [%]	Number of binucleated cells scored	Number of micrnucleated cells per 1000 bionucleate cells
Highly purified water (vehicle controls)
0	1.35	100	2000	3.5
Sodiu dihydrogen orthophosphate
125	1.36	103	2000	4.0
250	1.40	114	2000	3.0
500	1.48	139	2000	4.5
1000	1.38	109	2000	4.5
2000	1.36	205	2000	4.0
Mitomycin C
0.2	1.38	109	2000	37.5 s

24 -h exposure
Concentration of test item [µg/mL medium	CBPI	RI [%]	Number of binucleate cells scored	Number of micronucleated cells per 1000 binucleate cells
Highly purified water (vehicle control)
0	1.30	100	2000	3.5
Sodium dihydrogen orthophosphate
250	1.29	95	2000	3.0
500	1.31	104	2000	3.5
1000	1.26	87	2000	3.0
2000	1.26	87	2000	3.5
Colchicine
0.02	1.21	70	2000	53.5 s

CBPI = Cytokinesis block proliferation index

RI = Replicative Index

s. = significantly different from negative control (p ≤ 0.05)

Table 2: Experiment with metabolic activation (S9 mix)

4 -h exposure
Concentration of test item [µg/mL medium]	CBPI	RI [%]	Number of binucleate cells scored	Number of micronucleated cells per 1000 binucleate cells
Highly purified water (vehicle contol)
0	1.31	100	2000	3.5
Sodium dihydrogen orthophosphate
125	1.36	118	2000	3.5
250	1.29	94	2000	2.5
500	1.29	96	2000	4.5
1000	1.31	103	2000	3.5
2000	1.29	94	2000	3.5
Cyclophospamide
20	1.29	93	2000	27.0 s

CBPI = Cytokinesis block proliferation index

RI = Replicative Index

s. = significantly different from negative control (p ≤ 0.05)

Table 3: Experiment without metabolic activation (S9 mix) - 4 -hr exposure

Culture number	Concentration [µg/mL medium]	mononucleate	Number of binucleate cells#	multinucleate	CBPI	RI [%]	Number of binucleate cells scored	Number of micronucleated cells per 1000 binucleate cells	Significance chi^2 -test
Highly purified water (vehicle control)
1	0	345	144	11	1.33	100	1000	3	-
9	0	330	162	8	1.36	100	1000	4	-
Sodium dihydrogen orthophosphate
6	125	340	155	5	1.33	100	1000	4	n.s.
14	125	320	169	11	1.38	106	1000	4	n.s.
5	250	342	146	12	1.34	103	1000	3	n.s.
13	250	296	181	23	1.45	125	1000	3	n.s.
4	500	246	237	17	1.54	164	1000	5	n.s.
12	500	306	183	11	1.41	114	1000	4	n.s.
3	1000	344	143	13	1.34	103	1000	4	n.s.
11	1000	310	177	13	1.41	114	1000	5	n.s.
2	2000	334	160	6	1.34	103	1000	4	n.s.
10	2000	320	172	8	1.38	106	1000	4	n.s.
Mitmycin C
7	0.2	309	183	8	1.40	121	1000	36	s.
15	0.2	326	171	3	135	97	1000	39	s.

n.s. = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI = Replicative test

Table 4: Experiment without metabolic activation (S9 mix) - 24hr exposure

Culture number	Concentration [µg/mL medium]	mononucleate	Number of binucleate cells#	multinucleate	CBPI	RI [%]	Number of binucleate cells scored	Number of micronucleated cells per 1000 binucleate cells	Significane chi^2-test
Highly ourified water (vehicle control)
1	0	354	134	12	1.32	100	1000	4	-
9	0	378	106	16	1.28	100	1000	3	-
Sodium dihydrogen orthophosphate
5	250	366	105	29	1.33	103	1000	4	n.s.
13	250	379	120	1	1.24	86	1000	2	n.s.
4	500	363	122	15	1.30	94	1000	5	n.s.
12	500	356	130	14	1.32	114	1000	2	n.s.
3	1000	377	110	13	1.27	84	1000	3	n.s.
11	1000	382	112	6	1.25	89	1000	3	n.s.
2	2000	370	119	11	1.28	88	1000	4	n.s.
10	2000	389	103	8	1.24	86	1000	3	n.s.
Colchicine
7	0.02	403	86	11	1.22	69	1000	61	s.
15	0.02	408	85	7	1.20	71	1000	46	s.

n.s = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI = Replicative Index

Table 5: Experiment with metabolic activation (+S9 mix) -4hr exposure

Culture number	Concentration [µg/mL medium]	mononucleate	Number of binucleate cells#	multinucleate	CBPI	RI [%]	Number of binucleate cells scored	Number of micronucleated cells per 1000 binucleate cells	Significance chi^2-test
Highly purified water (vehicle control)
1	0	366	128	6	1.28	100	1000	4	-
9	0	343	143	14	1.34	100	1000	3	-
Sodium dihydrogen orthphosphate
6	125	320	165	15	1.39	139	1000	4	n.s.
14	125	345	146	9	1.33	97	1000	3	n.s.
5	250	367	127	6	1.28	100	1000	2	n.s.
13	250	362	127	11	1.30	88	1000	3	n.s
4	500	344	144	12	1.34	121	1000	4	n.s.
12	500	288	103	9	1.24	71	1000	5	n.s.
3	1000	325	163	12	1.37	132	1000	3	n.s.
11	1000	380	116	4	1.25	74	1000	4	n.s.
2	2000	378	106	16	1.28	100	1000	2	n.s.
10	2000	361	130	9	1.30	88	1000	5	n.s.
Cyclophosphamide
7	20	367	127	6	1.28	100	1000	26	s.
15	20	362	132	6	1.29	85	1000	28	s.

n.s. = not significantly different from negative control (p ≤ 0.05)

s. = significantly different from negative control (p ≤ 0.05)

CBPI = Cytokinesis block proliferation index

RI replicative index

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, sodium dihydrogenorthophosphate tested up to a concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.

Executive summary:

Test samples of Sodium dihydrogen orthophosphate were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

The test item was completely dissolved in highly purified water. The vehicle highly purified water served as the negative control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 μg Sodium dihydrogen orthophosphate/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 μg/mL medium.

Hence, 2000 μg/mL were employed as the top concentration for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure).

No signs of cytotoxicity were noted in the main study up to the top concentration of 2000 μg Sodium dihydrogen orthophosphate/mL medium in the experiments without and with metabolic activation.

Mitomycin C (at 0.2 μg/mL) and colchicine (at 0.02 μg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 μg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 125, 250, 500, 1000 and 2000 or 250, 500, 1000 and 2000 μg Sodium dihydrogen orthophosphate/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 3.0 to 4.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 μg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a frequency of 3.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure was observed. The vehicle result was within the historical control ranges.

In the positive control cultures the micronucleus frequencies were increased to 37.5 or 53.5 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 125, 250, 500, 1000 and 2000 μg Sodium dihydrogen orthophosphate/ml medium (4-h exposure) in the presence of metabolic activation ranged from 2.5 to 4.5 micronucleated cells per 1000 bi nucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 μg/ml medium. The frequency of

micronucleated cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 3.5 micronucleated cells per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges.

In the positive control culture the micronucleus frequency was increased to 27 .0 micronucleated cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.

Conclusion

Under the present test conditions, Sodium dihydrogen orthophosphate tested up to a concentration of 2000 μg/ml medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.

The results for the vehicle controls were within historical control range.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.