Barium bis(dihydrogenorthophosphate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

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:

Barium bis(dihydrogenorthophosphate)

EC Number:

236-715-1

EC Name:

Barium bis(dihydrogenorthophosphate)

Cas Number:

13466-20-1

Molecular formula:

Ba(H2PO4)2

IUPAC Name:

barium bis(dihydrogenorthophosphate)

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:

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

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 3160, 5000 µg/plate
concentrations used up to maximum recommended test concentration

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.05M HCl solution
- Justification for choice of solvent/vehicle: not specified

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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 (TA 100, TA 1535 with S9 mix)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the number of colonies by more than 50% compared to the vehicle control and/or 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 TA 98, TA 100, TA1535 and TA 1537 and 1.5-fold of the solvent control for TA 102 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.

Results and discussion

Test results

Key result

Species / strain:

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

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:

RANGE-FINDING/SCREENING STUDIES: Barium bis(dihydrogenorthophosphate) was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 μg Barium bis(dihydrogenorthophosphate)/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 3
- Negative (solvent/vehicle) historical control data: see table 3

Any other information on results incl. tables

Table 1: Plate incorporation test – number of reverted colonies (mean±standard deviation)
EXPERIMENT 1 (Plate incorporation test)
S9-Mix	Without
Test item (µg/plate)	TA98	TA100	TA102	TA1535	TA1537
NC	32.7±4.2	157.3±15.0	275.3±16.9	14.7±0.6	6.3±1.2
31.6	36.3±3.8	142.3±10.8	275.7±11.0	21.3±1.5	4.7±0.6
100	35.0±5.6	126.3±8.1	278.3±16.0	17.7±4.7	6.0±3.5
316	34.7±5.9	132.0±20.3	287.0±3.6	18.7±6.7	6.0±2.6
1000	31.7±4.9	152.0±25.4	287.7±8.3	19.7±8.3	4.7±0.6
3160	31.7±6.8	141.7±11.0	291.3±12.7	17.0±2.6	6.3±0.6
5000	34.2±2.1	131.0±9.2	270.0±16.7	16.3±7.8	4.3±2.1
Positive reference item	2-Nitrofluorene 10 µg/plate	Sodium azide 10 µg/plate	Mitomycin C 10 µg/plate	Sodium azide 10 µg/plate	9-Aminoacridine 100 µg/plate
	168.3±4.7	979.3±2.3	1025.3±83.5	153.0±4.0	82.0±5.6
S9-Mix	With
Test item (µg/plate)	TA98	TA100	TA102	TA1535	TA1537
NC	26.0±0.0	156.7±9.3	308.7±2.1	19.7±2.9	6.7±2.3
31.6	27.3±2.1	154.0±3.0	262.7±7.5	21.0±2.0	6.3±2.3
100	34.0±4.6	147.0±17.3	266.0±3.6	18.3±4.5	7.3±2.3
316	24.7±0.6	149.3±16.6	267.0±2.6	17.0±3.6	8.3±1.2
1000	31.3±4.2	129.0±23.5	264.3±4.9	19.7±5.5	8.3±1.5
3160	31.3±2.9	133.7-12.4	269.0±4.4	22.3±7.0	5.0±2.6
5000	40.0±10.1	111.3±4.0	271.3±1.2	19.7±5.0	5.3±2.3
Positive reference item	Benzo(a)pyrene 10 µg/plate	2-Aminoanthracene 2 µg/plate	Benzo(a)pyrene 10 µg/plate	2-Aminoanthracene 2 µg/plate	Benzo(a)pyrene 10 µg/plate
	157.0±3.0	975.7±10.7	1018.7±63.7	157.7±6.7	111.3±10.7
NC = Vehicle Control Table 2: Preincubation test – number of reverted colonies (mean±standard deviation) EXPERIMENT 2 (Preincubation test) S9-Mix Without   Test item (µg/plate) TA98 TA100 TA102 TA1535 TA1537 NC 37.0±2.0 154.0±12.5 295.0±2.6 22.3±2.1 7.3±1.2 31.6 35.3±3.1 143.3±15.6 260.0±1.0 19.0±6.1 7.3±2.1 100 31.7±7.1 164.7±7.8 273.0±19.1 22.7±4.7 7.0±0.0 316 31.3±3.1 135.7±23.8 292.3±1.2 18.7±2.5 7.0±1.7 1000 35.7±1.5 133.7±17.2 292.0±4.0 21.0±3.6 4.0±0.0 3160 31.0±5.2 146.7±11.2 282.7±4.2 24.0±3.6 8.3±1.2 5000 31.7±6.1 129.3±8.5 272.3±2.9 21.0±3.6 8.0±1.7 Positive reference item 2-Nitrofluorene 10 µg/plate Sodium azide 10 µg/plate Mitomycin C 10 µg/plate Sodium azide 10 µg/plate 9-Aminoacridine 100 µg/plate   173.7±6.5 951.3±4.5 1041.7±22.2 158.7±2.3 84.0±16.5             S9-Mix With               Test item (µg/plate) TA98 TA100 TA102 TA1535 TA1537 NC 30.3±2.3 162.3±10.0 286.0±8.7 20.0±8.2 7.7±1.2 31.6 31.3±1.2 162.3±9.5 279.7±18.8 17.7±3.8 5.0±1.0 100 35.3±5.0 145.7±9.1 279.3±5.7 20.0±5.3 6.0±0.0 316 35.3±0.6 182.3±3.2 268.0±20.3 18.0±3.0 7.0±3.0 1000 34.0±2.6 160.3±2.1 289.7±6.1 16.7±1.2 4.7±0.6 3160 30.7±6.4 173.7±9.5 279.0±13.2 18.7±4.9 5.7±2.1 5000 36.3±1.2 161.3±16.9 272.7±20.0 14.0±1.0 5.7±3.8 Positive reference item Benzo(a)pyrene 10 µg/plate 2-Aminoanthracene 2 µg/plate Benzo(a)pyrene 10 µg/plate 2-Aminoanthracene 2 µg/plate Benzo(a)pyrene 10 µg/plate   162.3±3.8 948.7±2.5 988.0±64.3 152.7±13.4 79.0±2.0 NC = Vehicle Control Table 3: History profile of negative and positive values of the years 2013 to 2015 (n=83 studies) Strain S9 mix   mean SD Min Max Negative Reference item TA 98 -   29.9 5.7 19 49 +   31.3 5.9 14 49 TA100 -   148.5 18.1 103 191 +   149.2 18.2 106 195 TA102 -   275.5 15.5 240 319 +   279.8 16.2 245 319 TA1535 -   19.4 4.4 10 34 +   19.2 4.4 8 42 TA1537 -   6.4 1.8 2 10 +   6.5 1.9 0 10 Positive Reference Item TA 98 - 2-nitrofluorene 148.4 33.6 91 305 + Benzo(a)pyrene 147.3 33.7 91 315 TA100 - Sodium azide 928.7 107.8 463 1209 + 2-aminoanthracene 937.9 107.8 463 1209 TA102 - Mitomycin C 1014.6 121.4 756 1637 + Benzo(a)pyrene 1008.9 109.5 757 1571 TA1535 - Sodium azide 133.2 28.9 51 220 + 2-aminoanthracene 133.3 28.5 49 271 TA1537 - 9-aminoacridine 71.1 28.8 26 178 + Benzo(a)pyrene 81.5 28.9 28 189

Applicant's summary and conclusion

Conclusions:

Under the test conditions of this study the test substance was not mutagenic in bacteria with and without metabolic activation.