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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-12-15 to 2016-01-14
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2014-05-14
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Triiron bis(orthophosphate)
EC Number:
239-018-0
EC Name:
Triiron bis(orthophosphate)
Cas Number:
14940-41-1
Molecular formula:
Fe.2/3H3O4P or Fe3(PO4)2
IUPAC Name:
tris(lambda2-iron(2+)) diphosphate
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: light grey powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10°C to +25°C, in a tightly closed container and store in a cool, dry and well-ventilated place.

Method

Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
TA102: his G428
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:
S9-mix (containing 5 % S9): 0.4 M MgCl2 + 1.65 M KCl salt solution (2 mL); glucose-6-phosphate (141.0 mg); NADP (306.5 mg; phosphate buffer (pH 7.4; 50 mL); fill up to a total of 100.0 mL with sterile aqua ad iniectabilia
Test concentrations with justification for top dose:
Plate incorporation test: 31.6, 100, 316, 1000, 3160, and 5000 µg/plate (with and without metabolic activation)
Preincubation test: 31.6, 100, 316, 1000, 3160, and 5000 µg/plate (with and without metabolic activation)
The test concentrations were chosed based on a preliminary cytotoxicity test, which indicted no cytotoxicity up to a concentration of 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 0.05 M HCl solution
- Justification for choice of solvent/vehicle: the test item was not soluble in any of the solvents recommended: water, dimethylsulfoxide (DMSO), ethanol or acetone. The test item was completely soluble at 100 μg/mL 0.05 M HCl solution. Hence, the test item was suspended in 0.05 M HCl solution for concentrations of 31.6, 100, 316, 1000, 3160, or 5000 μg/plate. For concentrations lower than 10.0 μg/plate, which were used in the preliminary cytotoxicity test, the test item was completely dissolved. Fresh preparations of the test item were used for the treatment in all experimental parts.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.05 M HCl solution
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation; independent experiment 1) and preincubation (independent experiment 2)

MAIN EXPERIMENT (two independent experiments with and without metabolic activation)
1) plate incorporation method (independent experiment 1)
2 mL of top agar were distributed into culture tubes. 0.1 mL of Salmonella cell suspension (containing approximately 10^8 viable cells in the late exponential or early stationary phase) 0.1 mL of test item (or 0.1 mL solvent or 0.1 mL positive control) and 0.5 mL of S9 mix were added to these culture tubes. In the assay without metabolic activation, the S9 mix was substituted with 0.5 mL phosphate buffer.
The test components were mixed and then poured onto a minimal glucose agar plate (Minimal Glucose Agar medium E).
The plates were inverted and placed in a dark 37°C incubator for 48 to 72 hours. The revertant colonies on the test plates and on the control plates were counted with a colony counter.

2) Preincubation method (independent experiment 2)
The test item was preincubated with the test strain (containing approximately 10^8 viable cells in the late exponential or early stationary phase) and sterile buffer (0.5 mL) or the metabolic activation system (0.5 mL) for 20 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. 0.1 mL of test item (or 0.1 mL solvent or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker. The remaining steps were the same as described for the plate incorporation method.

NUMBER OF REPLICATIONS: 3 replicates for each experimental point

DETERMINATION OF CYTOTOXICITY
Prior to the main test two preliminary cytotoxicity tests (plate incorporation test, with and without metabolic activation) were carried out in test strain TA100. The following concentrations were tested: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160, and 5000 µg/plate
Rationale for test conditions:
The test concentrations were chosen based on a preliminary cytotoxicity test, which indicted no cytotoxicity up to a concentration of 5000 µg/plate. Precipitation was observed for a concentration of 1000 µg/plate and higher.
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:
U-test according to MANN and WHITNEY and Spearman’s rank correlation coefficient

Results and discussion

Test results
Key result
Species / strain:
other: TA98, TA100, TA102, TA1535, and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
please refer to the field "Additional information on results" below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- test item precipitation was noted at concentrations of 1000 to 5000 μg/plate in both experiments.
- no signs of cytotoxicity were noted.

MAIN STUDY (2 independent experiments)
1) Test-specific confounding factors:
- Precipitation: test item precipitation was noted in both experiments, each carried out without and with metabolic activation, at concentrations of 1000 μg/plate and higher in all test strains.

2) Cytotoxicity:
- cytotoxicity (reduction of the number of revertants by more than 50%) was noted in both experiments with metabolic activation in test strain TA1537 at 5000 μg/plate.

3) Genotoxicity:
- no increase in revertant colony numbers as compared with control counts was observed for the test item in any of the two independent experiments without and with metabolic activation.
- positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Please also refer to the field "Attached background material" below.

HISTORICAL CONTROL DATA
- Positive historical control data / Negative (solvent/vehicle) historical control data: please refer to Table 1 in the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: History profile of negative and positive control values of the years 2013 to 2015 (n=83 studies) - Data obtained from plate incorporation and preincubation tests

Negative Reference Item

Strain

S9-Mix

TA 98

TA 100

TA 102

TA 1535

TA 1537

-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

S9-Mix

TA 98

TA 100

TA 102

TA 1535

TA 1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

2-nitrofluorene

Benzo [a] pyrene

Sodium azide

2-aminoanthracene

Mitomycin C

Benzo [a] pyrene

Sodium azide

2-amino-anthracene

9-amino-acridine

Benzo [a] 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

 

 

 

Applicant's summary and conclusion

Conclusions:
The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.