Dicalcium pyrophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:

Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. Coli WP2 uvrA were all negative with and without metabolic activation (OECD 471)

Cytogenicity (micronucleus assay): negative (OECD 487)

Gene mutation (mammalian cells / TK test): negative (OECD 476)

In vivo:

no data available and no further data needed

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:

25 Feb - 23 Jun 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium), trp operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: distilled water was used as solvent/vehicle, since this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene (10-50 µg/plate)

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other: daunomycin (6 µg/plate); ICR 191 acridine (1 µg/plate)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

INITIAL TEST
METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: 3

CONFIRMATORY TEST
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies on the test plates compared to the solvent/vehicle control plates

Evaluation criteria:

In general, a 2 or 2.5-fold increase in the number of revertant colonies per plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials. The presence of dose response is a further criterion for mutagenic materials.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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 examined

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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitation was seen at all test material concentrations, which did not influence the results of the assay

RANGE-FINDING/SCREENING STUDIES: A preliminary experiment was conducted to determine cytotoxicity and solubility of the test substance. Starting with the test material concentration of 5 mg/plate (recommended by OECD GL 471) and subsequent serial dilutions (ratio 1:2) following concentrations of test material were applied: 5, 2.5, 1.25, 0.625, 0.313 and 0.156 mg/plate on two bacteria strains (TA 98 and TA 1535) with different mutation type (frameshift and base-pair substitution) with and without metabolic activation.
The highest concentration of that solution was chosen for the main test that caused low cytotoxicity and/or where the precipitate did not interfere with the scoring according to OECD GL 471.

COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control values were acceptable and compatible with the historical control values.

Test Material concentrations:

Concentration 1: 5 mg/plate

Concentration 2: 2.5 mg/plate

Concentration 3: 1.25 mg/plate

Concentration 4: 0.625 mg/plate

Concentration 5: 0.313 mg/plate

 

 

Table 2:Mean plate counts and standard deviations (SD); strain: TA98

 

Controls/ test substance conc.	Colony count
	TA98
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	18	2	13	2
NK+S9	23	1	20	2
PK	508	36	632	39
PK1+S9	2154	207	2032	116
PK2+S9	139	20	160	11
Conc. 1	18	4	14	2
Conc. 1+S9	21	2	23	4
Conc. 2	17	2	12	4
Conc. 2+S9	25	5	24	7
Conc. 3	20	3	16	8
Conc. 3+S9	30	4	16	3
Conc. 4	15	5	15	1
Conc. 4+S9	25	6	18	1
Conc. 5	22	4	12	3
Conc. 5+S9	27	6	23	3

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (daunomycin)

PK1 - positive control 1 with S9 (2-aminoanthracene)

PK2 - positive control 2 with S9 (benzo(a)pyrene)

Conc. - concentration

 

 

Table 3:Mean plate counts and standard deviations (SD); strain: TA100

 

Controls/ test substance conc.	Colony count
	TA100
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	82	1	87	16
NK+S9	100	10	96	4
PK	508	52	775	202
PK1+S9	1966	109	1783	111
PK2+S9	686	77	724	95
Conc. 1	87	8	84	7
Conc. 1+S9	100	8	85	12
Conc. 2	100	10	79	11
Conc. 2+S9	103	5	93	8
Conc. 3	82	6	80	6
Conc. 3+S9	103	7	88	9
Conc. 4	90	8	82	2
Conc. 4+S9	97	2	94	16
Conc. 5	97	4	79	3
Conc. 5+S9	109	17	89	10

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (sodium azide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

PK2 - positive control 2 with S9 (benzo(a)pyrene)

Conc. - concentration

 

 

Table 4:Mean plate counts and standard deviations (SD); strain: TA1535

 

Controls/ test substance conc.	Colony count
	TA1535
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	10	3	8	4
NK+S9	9	2	11	4
PK	375	33	312	28
PK1+S9	82	10	47	17
Conc. 1	8	2	8	3
Conc. 1+S9	9	2	9	7
Conc. 2	10	4	8	4
Conc. 2+S9	5	4	10	0
Conc. 3	11	8	14	3
Conc. 3+S9	5	1	8	3
Conc. 4	8	6	9	3
Conc. 4+S9	7	2	6	2
Conc. 5	10	3	11	3
Conc. 5+S9	8	2	9	1

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (sodium azide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

 

 

Table 5: Mean plate counts and standard deviations (SD); strain: TA1537

 

Controls/ test substance conc.	Colony count
	TA1537
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	7	2	4	3
NK+S9	7	3	7	4
PK	1635	439	122	15
PK1+S9	144	23	177	29
Conc. 1	7	4	6	3
Conc. 1+S9	6	5	5	1
Conc. 2	7	2	5	2
Conc. 2+S9	6	0	6	1
Conc. 3	6	1	7	1
Conc. 3+S9	10	2	6	1
Conc. 4	8	1	6	4
Conc. 4+S9	8	3	5	1
Conc. 5	6	3	3	2
Conc. 5+S9	10	2	7	5

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (ICR 191 acridine)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

 

 

Table 6:Mean plate counts and standard deviations (SD); strain: WP2uvrA

 

Controls/ test substance conc.	Colony count
	WP2uvrA
	Main test: Preincubation method		Confirmatory test: Plate incorporation method
	Mean	SD	Mean	SD
NK	17	2	27	4
NK+S9	13	2	31	4
PK	1000	30	1350	38
PK1+S9	402	31	357	44
Conc. 1	18	3	35	2
Conc. 1+S9	16	8	35	4
Conc. 2	17	5	35	3
Conc. 2+S9	16	4	29	2
Conc. 3	18	6	25	3
Conc. 3+S9	17	8	32	2
Conc. 4	16	1	26	5
Conc. 4+S9	16	1	36	11
Conc. 5	20	8	34	2
Conc. 5+S9	17	6	35	5

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (4-nitroquinoline-1-oxide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

Conclusions:

Under the conditions of this study, the test material did not induce reverse gene mutations in Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) or Escherichia coli (WP2 uvrA) tester strains. Therefore, the test material is considered to be not mutagenic in bacteria.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Feb - 28 Apr 2015

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus (tk1)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10 % [v/v] heat inactivated horse serum (HS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

0.625, 1.25, 2.5 and 5 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Cell culture medium (RPMI 1640) containing 10 % [v/v] HS with and without metabolic activation (S9), respectively was used as solvent/vehicle.
- Justification for choice of solvent/vehicle: this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 (± 0.1) h in the main test, 3.5 (± 0.1) h in the confirmatory test
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 11-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-16 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT) at a final concentration of 3 μg/mL

NUMBER OF REPLICATIONS: 2 each in the main and confirmatory test

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The cloning efficiency (CE) of cells and the spontaneous mutation frequency (MF) of negative controls should be within their limit values (CE ≥ 50 %; MF (negative control) 50-300 x 10-6). The MF of positive controls should be increased 2 times or more than MF in corresponding negative controls in each experiment.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH during the exposure of the cells to the test substance did not change (colour of the pH indicator in the culture medium).
- Precipitation: Slight precipitations were seen in all test material concentrations which did not influence the results of the assay.

RANGE-FINDING/SCREENING STUDIES:
A preliminary experiment was conducted to determine cytotoxicity and solubility of the test substance. Starting with the test material concentration of 5 mg/mL (recommended by OECD GL 476) and subsequent serial dilutions (ratio 1:5) following concentrations of test material were applied: 5 mg/mL, 1 mg/mL, 0.2 mg/mL, 0.04 mg/mL, 0.008 mg/mL, 0.0016 mg/mL and 0.00032 mg/mL. Cell culture medium containing 10 % [v/v] HS was used as solvent.
The vessels were shaken well to keep the particles homogenous in solution before next dilution of test specimen suspension was done.
The highest concentration (5 mg/mL) of that solution was chosen for the main test, which showed low cytotoxicity (Table 3) and where the precipitate did not interfere with the scoring according to OECD GL 476.
Four analysable concentrations (separation factor 2) were applied in the main test as well as the negative control (duplicates) and single positive controls with and without metabolic activation (S9), respectively.
The concentrations of test compound applied in the main test were thus:
Concentration 1 : 5 mg/ml
Concentration 2 : 2.5 mg/ml
Concentration 3 : 1.25 mg/ml
Concentration 4 : 0.625 mg/ml

COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control values were acceptable and compatible with the historical control values.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
At all test concentrations, negative and positive controls (with and without metabolic activation (S9-mix)) the cloning efficiency was within the limit.
No relevant signs of toxicity were determined within the test material concentrations (Table 1).

Table 1. Mean cloning efficiency (CE1 and CE2) in the main and confirmatory tests.

Test item	CE1	CE2
Main test
Negative control (-S9)	1.00	0.97
Negative control (+S9)	0.86	0.70
Positive control (-S9)	0.70	0.73
Positive control (+S9)	0.62	0.51
5 mg/mL (-S9)	0.58	0.95
5 mg/mL (+S9)	0.74	0.84
2.5 mg/mL (-S9)	0.62	1.10
2.5 mg/mL (+S9)	0.62	0.85
1.25 mg/mL (-S9)	0.77	0.79
1.25 mg/mL (+S9)	0.57	0.81
0.625 mg/mL (-S9)	0.79	0.85
0.625 mg/mL (+S9)	0.92	0.82
Confirmatory test
Negative control (-S9)	0.82	0.85
Negative control (+S9)	0.92	0.91
Positive control (-S9)	0.65	0.68
Positive control (+S9)	0.60	0.56
5 mg/mL (-S9)	0.83	0.95
5 mg/mL (+S9)	0.84	0.87
2.5 mg/mL (-S9)	0.60	0.83
2.5 mg/mL (+S9)	0.87	0.75
1.25 mg/mL (-S9)	0.66	0.78
1.25 mg/mL (+S9)	1.01	0.77
0.625 mg/mL (-S9)	0.69	0.80
0.625 mg/mL (+S9)	1.03	0.97

 

Table 2. Mutation frequencies (MF) for big and small colonies in the main and confirmatory tests.

Test item	MF Big colonies	MFG Small conolines
Main test
Negative control (-S9)	104.25	53.50
Negative control (+S9)	161.64	77.96
Positive control (-S9)	227.14	660.42
Positive control (+S9)	529.18	871.87
5 mg/mL (-S9)	101.12	65.78
5 mg/mL (+S9)	112.12	89.21
2.5 mg/mL (-S9)	113.96	58.08
2.5 mg/mL (+S9)	127.84	49.63
1.25 mg/mL (-S9)	115.43	60.34
1.25 mg/mL (+S9)	155.45	60.65
0.625 mg/mL (-S9)	126.65	40.09
0.625 mg/mL (+S9)	129.28	58.30
Confirmatory test
Negative control (-S9)	101.05	65.82
Negative control (+S9)	91.28	36.06
Positive control (-S9)	249.29	788.72
Positive control (+S9)	240.58	361.75
5 mg/mL (-S9)	101.91	77.49
5 mg/mL (+S9)	80.73	47.37
2.5 mg/mL (-S9)	69.20	73.42
2.5 mg/mL (+S9)	74.05	47.69
1.25 mg/mL (-S9)	63.90	94.05
1.25 mg/mL (+S9)	65.67	34.77
0.625 mg/mL (-S9)	42.70	68.49
0.625 mg/mL (+S9)	70.06	28.91

Conclusions:

Under the conditions of this study, the test material did not induce a significant increase in mutation frequencies in mouse lymphoma L5178Y cells. Therefore, the test material is considered to be not mutagenic in mammalian cells.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24. Feb. 2015 - 20. March 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The duration of the extended treatment was not specified further than lasting 1.5 - 2 cell cycles, no historical vehicle and positive control data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted Sep 2014

Deviations:

yes

Remarks:

the duration of the extended treatment was not specified further than lasting 1.5 - 2 cell cycles, historical data is not given

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 supplemented with fetal bovine serum (FBS) to a final concentration of 10% (v/v)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

4 h treatment (with and without metabolic activation): 0.36, 1.07 and 3.2 µg/mL
Extended treatment (no further information on duration, without metabolic activation):0.36, 1.07 and 3.2 µg/mL

3.2 µg/mL was used as highest dose because the solulibity was the limiting factor.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: cholchicine 0.2 µg/mL without S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h and 1.5 - 2 cell cycles, no further information available (the cell cycle duration was determined in negative control cultures, the duration was not reported in the study report)
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment and extended treatment: after 1.5 - 2 cell cycles, no further information available (the cell cycle duration was determined in negative control cultures, the duration was not reported in the study report

STAIN (for cytogenetic assays): Hoechst 33258 solution and Pyronin Y

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: At least 2000 (1000 cells per culture, two cultures per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC). Relative cytotoxicity = 100% - RICC (%).

OTHER EXAMINATIONS:
- Other: in order to estimate the required cell cycle number and treatment duration (1.5 - 2 cell cycles after treatment start), additional vehicle control cultures were maintained. Two of these cultures were harvested immediately after removing the test substance from the treated cultures and the cells were counted. Approximately 24 h later two untreated cell cultures were harvested for cell counting. Mitosis was shown by cell counting at the end of analysis and comparing this value with the number of cells at the beginning of the treatment.

Evaluation criteria:

A test substance was considered to be genotoxic if at least a 3-fold increase in number of micronuclei in relation to the negative control was counted and/or a dose-response relationship was observed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitation was observed at and above 3.2 µg/mL.

RANGE-FINDING/SCREENING STUDIES: A range-finding study was performed, using the concentrations 0.64, 3.2, 16, 80 and 400 µg/mL. Due to precipitation in the culture medium, 3.2 µg/mL was selected as the highest concentration in the main study (see Table 1 under 'Any other information on results incl. tables').

COMPARISON WITH HISTORICAL CONTROL DATA: No.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The cytotoxicity was estimated to be 12% at the highest concentration of 3.2 µg/mL.

Table 1: Results of main experiment

Test item	Concentration [µg/mL]	Threshold value*	Number of cells with MN (average of 2 cultures)
Exposure period 3h, fixation time after 1.5 – cell cycles**, without S9 mix
Vehicle control	-	12.0	4.0
CH	-	12.0	23.0
Test substance	0.36	12.0	4.5
	1.07	12.0	4.5
	3.2	12.0	9.0
Exposure period 3h, fixation time after 1.5 – cell cycles**, with S9 mix
Vehicle control	-	25.5	8.5
B[a]P	-	25.5	27.0
Test substance	0.36	25.5	5.0
	1.07	25.5	4.5
	3.2	25.5	14.0
Exposure period 1.5 - 2 cell cycles, fixation time after 1.5 – 2 cell cycles**, without S9 mix
Vehicle control	-	13.5	4.5
CH	-	13.5	18.0
Test substance	0.36	13.5	6.5
	1.07	13.5	6.5
	3.2	13.5	10.5

CH: colchicine; B[a]P: benzo[a]pyrene (positive controls)

*the 3-fold increase in micronuclei in comparison with the average

micronuclei in the negative control

Conclusions:

The test material did not induce any toxicologically significant increases in the number of cells with micronuclei and is therefore considered to be non-clastogenic and non-aneugenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Since all in vitro genetic toxicity tests were negative no further in vivo testing is required according to Annex VIII (8.4) of the REACh regulation.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity of calcium bis(ortho)phosphate was evaluated in three different in vitro tests.

Genetic toxicity (mutagenicity) in bacteria in vitro

A reliable bacterial gene mutation study (Ames Test) performed according to OECD TG 471 and in compliance with GLP is available with dicalcium pyrophosphate (CAS 7790-76-3). The Salmonella typhimurium tester strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA were tested according to the pre-incubation (experiment I) and plate incorporation (experiment II) procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix ). Two independent experiments were conducted in the concentration range from 313 to 5000 µg/plate (experiment I and II). No relevant cytotoxicity was observed in a preliminary toxicity test performed with tester strains TA 98 and TA 1535 after treatment with the test item up to 5000 µg/plate in the absence and presence of metabolic activation. Slight precipitation was seen in all test material concentrations which did not influence the results of the assay. Appropriate solvent (distilled water) and positive controls were included and gave the expected results. No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation. Therefore, the test material was considered to be non-mutagenic under the conditions of the test.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro Micronucleus Test equivalent or similar to OECD TG 487 and in compliance with GLP was performed with dicalcium pyrophosphate (CAS 7790-76-3). Chinese hamster lung fibroblasts (V79) were treated in duplicate cultures with the test material or vehicle (RPMI 1640 medium supplemented with 10% (v/v) FBS) in the absence or presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). Short-term (4 hours with and without S9 mix) and long-term (1.5 to 2 cell cycles without S9 mix) experiments were conducted at concentrations of 0.36, 1.07 and 3.2 µg/mL. Fixation of the cells was performed 24 hours (approx. 1.5 to 2.0 normal cell cycles) after start of exposure with the test material. Appropriate solvent and positive controls were included in the test and gave the expected results. The number of micronucleated cells found after treatment with the test item was within the normal range of the negative control and thus no genotoxic effects were recorded either in the presence or in the absence of metabolic activation.Slight precipitation was observed at 3.2 µg/mL. The cytotoxicity was estimated to be 12% at the highest concentration of 3.2 µg/mL. Based on the results of the study the test material is considered not to be clastogenic or aneugenic under the conditions of this in vitro study.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

An in vitro Mammalian Cell Gene Mutation Test was performed with dicalcium pyrophosphate in mouse lymphoma L5178Y cells (heterozygous at the thymidine kinase locus) according to OECD TG 476 and under GLP. The cells were treated with the test substance in duplicate cultures, together with vehicle (RPMI 1640 medium with 10% HS) and positive controls (single cultures). The cells were exposed to the test substance for 3 to 3.5 hours in the absence and presence of metabolic activation (1254 Aroclor-induced rat liver S9-mix) in the main and the confirmatory test. The concentration range of the test material in both experiments was 625 to 5000 µg/mL following the results of a preliminary toxicity test. Slight precipitations were seen in all test material concentrations which did not influence the results of the assay. The vehicle controls were within the normal range for the L5178Y cell line at the TK locus. The positive controls induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. Cytotoxicity was observed up to 5000 µg/mL. Thus, since the test material did not induce any toxicological significant increases in mutant frequencies up to the highest concentration of 5000 µg/mL it was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Since all in vitro test showed clear negative results it is considered scientifically unjustified to do any further testing to assess the genotoxicity and mutagenicity of dicalcium pyrophosphate in vivo. This is in line with Annex VIII (8.4) of the REACh regulation as only if there are positive results in any of the in vitro genotoxicity studies an appropriate in vivo mutagenicity study shall be considered.

Justification for classification or non-classification

The available data on genetic toxicity with dicalcium pyrophosphate (CAS 7790 -76 -3) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.