Dizinc pyrophosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jan 2016-18 Feb 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dizinc pyrophosphate

EC Number:

231-203-4

EC Name:

Dizinc pyrophosphate

Cas Number:

7446-26-6

Molecular formula:

H4O7P2.2Zn

IUPAC Name:

dizinc(2+) (phosphonooxy)phosphonate

Test material form:

solid: particulate/powder

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

First experiment (plate incorporation method): 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg/plate with and without metabolic activation
Second experiment (preincubation method): 31.6, 100, 316, 1000, 3160 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.05 M HCl
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the solvents recommended: water, dimethylsulfoxide (DMSO), ethanol or acetone, but was completely soluble at 3.16 mg/mL 0.05 M HCl in preliminary solution tests. For concentrations lower than or equal to 316 μg/plate, the test item was completely dissolved. For concentrations of 1000, 3160 and 5000 μg/plate the test material was suspended in 0.05 M HCl solution.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.05 M HCl

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 2 replications each in 2 independent experiements

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn. Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Evaluation criteria:

A test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537, and 1.5-fold of the solvent control for TA102 in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) is observed.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first. A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the top concentration of 5000 μg/plate in all test strains

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test material precipitation was noted in all experiments at concentrations of 3160 and 5000 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

Negative Reference Item
Strain TA 98 TA 100 TA 102 TA 1535 TA 1537
S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 29.9 31.3 148.5 149.2 275.5 279.8 19.4 19.2 6.4 6.5
SD 5.7 5.9 18.1 18.2 15.5 16.2 4.4 4.4 1.8 1.9
Min 19 14 103 106 240 245 10 8 2 0
Max 49 49 191 195 319 319 34 42 10 10


Positive Reference Item
Strain TA 98 TA 100 TA 102 TA 1535 TA 1537
S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
2-nitro- Benzo[a] Sodium 2-amino- Mitomycin Benzo[a] Sodium 2-amino- 9-amino- Benzo[a]
fluorene pyrene azide anthracene C pyrene azide anthracene acridine pyrene

Mean 148.4 147.3 928.7 937.9 1014.6 1008.9 133.2 133.3 81.1 81.5
SD 33.6 33.7 107.8 99.8 121.4 109.5 28.9 29.5 28.8 28.9
Min 91 91 463 703 756 757 51 49 26 28
Max 305 315 1209 1181 1637 1571 220 271 178 189

Any other information on results incl. tables

Dizinc pyrophosphate

EXPERIMENT 1(Standard Plate Test, SPT)
S9-Mix	Without
Test Item (mg/plate)	Base-pair substitution type			Frameshift type
	TA 100	TA 102	TA 1535	TA 98	TA 1537
VC	135.0±9.2	268.3±5.5	17.3±1.5	31.7±6.1	5.7±1.5
31.6	149.3±3.5	264.7±3.8	14.3±0.6	31.3±8.3	4.0±0.0
100	167.7±9.0	263.0±1.0	16.3±1.5	29.7±4.9	7.0±1.7
316	150.7±20.8	267.0±1.0	16.7±1.5	32.7±4.9	4.7±1.5
1000	126.3±7.6	276.7±9.5	15.0±1.0	32.7±1.5	6.7±3.5
3160	164.7±13.3	268.0±7.8	15.0±1.0	30.7±7.1	5.0±1.0
5000	17.3±4.0	21.7±1.5	6.3±1.5	9.0±1.0	1.3±0.6
PC (mg/plate)	NaN3(10)	MMC (10)	NaN3(10)	2NF (10)	AAC (100)
	916.3±10.7	1018.3±9.5	143.3±1.5	177.0±3.0	51.70±3.1
S9-Mix	With
Test Item (mg/plate)	Base-pair substitution type			Frameshift type
	TA100	TA 102	TA 1535	TA98	TA1537
VC	149.7±3.8	251.7±1.5	22.0±0.0	36.0±2.6	6.0±1.0
31.6	166.3±10.2	259.0±0.0	18.0±0.0	29.0±2.0	6.7±3.1
100	145.7±9.0	270.7±9.2	17.3±1.5	32.0±3.6	5.3±1.5
316	142.3±8.1	297.0±19.1	14.7±0.6	25.7±3.8	6.0±1.0
1000	134.7±7.6	253.7±5.5	16.0±2.6	27.3±6.0	5.7±1.5
3160	150.0±14.0	256.3±3.2	16.0±2.6	24.7±3.1	6.0±1.0
5000	27.3±5.7	24.7±6.4	5.7±1.2	8.0±1.0	1.0±0.0
PC (mg/plate)	AAN (2)	B[a]P (10)	AAN (2)	B[a]P (10)	B[a]P (10)
	878.0±37.6	1022.3±26.3	141.7±1.2	177.0±2.0	52.7±3.1

VC = Vehicle control; PC = Positive control

NaN₃= Sodium azide

2NF = 2-Nitrofluorene

MMC = Mitomycin C

AAC = 9-Aminoacridine

AAN = 2-Aminoanthracene

B[a]P = Benzo[a]pyrene

 

EXPERIMENT 2(Standard Plate Test with Incubation)
S9-Mix	Without
Test Item (mg/plate)	Base-pair substitution type			Frameshift type
	TA100	TA 102	TA 1535	TA98	TA1537
VC	145.0±20.0	281.7±23.5	21.7±8.1	23.7±2.1	8.3±0.6
31.6	155.7±4.9	254.7±1.2	20.0±1.7	28.3±1.5	6.3±0.6
100	141.0±21.1	254.3±2.1	18.3±0.6	27.0±3.5	5.7±1.5
316	138.3±15.5	282.3±22.8	16.0±1.7	27.7±3.2	8.3±1.2
1000	151.7±4.9	260.7±15.6	15.0±1.0	25.3±3.2	6.0±1.7
3160	135.7±12.4	268.0±2.6	22.3±5.9	26.3±1.2	6.3±0.6
5000	32.0±10.5	104.70±3.5	6.3±1.2	8.3±1.5	1.3±0.6
PC (mg/plate)	NaN3(10)	MMC (10)	NaN3(10)	2NF (10)	AAC (100)
	974.3±62.3	991.7±2.5	114.3±11.7	186.7±14.3	68.0±0.0
S9-Mix	With
Test Item (mg/plate)	Base-pair substitution type			Frameshift type
	TA100	TA 102	TA 1535	TA98	TA1537
VC	166.3±2.5	295.0±4.0	18.0±2.0	28.7±0.6	7.7±2.1
31.6	135.0±11.3	278.0±3.0	15.7±0.6	30.7±3.1	9.0±1.0
100	138.7±9.3	267.3±13.2	16.0±1.0	27.0±1.0	6.7±2.9
316	142.3±23.1	255.7±2.1	20.3±3.1	25.3±0.6	7.0±1.7
1000	126.3±14.5	270.0±26.9	15.7±0.6	25.7±6.0	7.3±2.5
3160	141.0±7.2	270.3±7.8	15.7±2.5	34.3±0.6	4.3±0.6
5000	32.3±3.5	93.3±2.1	7.3±1.2	8.3±0.6	1.7±0.6
PC (mg/plate)	AAN (2)	B[a]P (10)	AAN (2)	B[a]P (10)	B[a]P (10)
	935.0±69.4	1026.0±72.5	133.0±24.3	185.0±6.0	64.3±0.6

VC = Vehicle control; PC = Positive control

NaN₃= Sodium azide

2NF = 2-Nitrofluorene

MMC = Mitomycin C

AAC = 9-Aminoacridine

AAN = 2-Aminoanthracene

B[a]P = Benzo[a]pyrene

Applicant's summary and conclusion

Conclusions:

Interpretation of results: the test material dizinc pyrophosphate is negative for mutagenicity (non-mutagenic) with and without metabolic activation.