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Diss Factsheets

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:
key study
Study period:
2015-02-25 to 2015-06-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
historical control data incomplete (benzo(a)pyrene data missing; positive control data not clearly labelled); Preliminary results missing; strain charac. confirmations not fully described. Proficiency of the lab not comparable with other laboraotries.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimanganese bis(orthophosphate)
EC Number:
237-997-9
EC Name:
Trimanganese bis(orthophosphate)
Cas Number:
14154-09-7
Molecular formula:
Mn3O8P2
IUPAC Name:
trimanganese bis(orthophosphate)
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: pink solid powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature 15 - 25 °C

Method

Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp-
Species / strainopen allclose all
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:
S9-mix (containing 10 % S9)
Test concentrations with justification for top dose:
Main test (preincubation method): 0.313, 0.625, 1.25, 2.5, and 5 mg/plate (with and without metabolic activation)
Confirmatory test (plate incorporation method): 0.313, 0.625, 1.25, 2.5, and 5 mg/plate (with and without metabolic activation)
In preliminary tests the highest concentration was determined considering the criteria solubility and cytotoxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- 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 bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene / daunomycin
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR 191 acridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation (main test) and in agar (plate incorporation, confirmatory test);

MAIN TEST (preincubation with and without metabolic activation)
The test solution (0.1 mL) was preincubated with the test strain (0.1 mL, containing approx. 10E8 viable cells) and sterile buffer or the metabolic activation system (0.5 mL) for 20 minutes at 30 °C - 37 °C prior to mixing with the overlay agar (2.0) mL. Tubes were aerated during the pre-incubation by using a shaker.
The content of each tube was poured over the surface of a minimal agar plate. This overlay agar was allowed to solidify before incubation.
After a period of incubation (about 48 - 72 hours, 37 ± 1 °C) revertant colonies were counted and compared with the number of spontaneous revertants in an untreated and/or solvent control culture.
The bacterial colonies were counted using the photo system "Argus X1/Total Lab-software".

CONFIRMATORY TEST (plate incorporation with and without metabolic activation)
For the test without metabolic activation 0.1 mL of the test solutions, 0.1 mL of fresh bacterial culture and 0.5 mL of sterile buffer were mixed with the overlay agar.
For the assay with metabolic activation 0.5 mL of S9 mixture was mixed with the overlay agar (2.0 mL), together with the bacteria and test solution.
The content of each tube was mixed and poured over the surface of a minimal agar plate.
This overlay agar was allowed to solidify before incubation.
After a period of incubation (about 48 - 72 hours, 37 ± 1 °C) revertant colonies were counted and compared with the number of spontaneous revertants in an untreated and/or solvent control culture.

NUMBER OF REPLICATIONS: triplicate plating was used at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
A preliminary experiment was conducted to determine cytotoxicity and solubility of the test substance. Starting with the test material concentration of 5 mg/plate 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 471.
Rationale for test conditions:
A preliminary experiment was conducted to determine cytotoxicity and solubility of the test substance. 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 471.
Evaluation criteria:
A 2 or 2.5-fold increase in the number of revertant colonies/plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials (MOLTOX Salmonella Mutagenicity Test Kit Instruction Manual, 2014). The presence of dose response is a further criterion for mutagenic materials.
Statistics:
Means of individual plate counts (triplicates) were calculated for test solutions and controls.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitations were seen in all test material concentrations which did not influeunce the results of the assay.

MAIN TEST (preincubation)
No genotoxic activity caused by the test material was detected under the described test conditions. In addition, a presence of test dose response was not detected.
Please also refer to the field "Attached background material" below.

CONFIRMATORY TEST (plate incorporation)
No genotoxic activity caused by the test material was detected under the described test conditions. In addition, a presence of test dose response was not detected.
Please also refer to the field "Attached background material" below.

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:
The colony counts of negative and positive controls are in the dimensions of historical data and fulfil the requirements according to the MOLTOX Salmonella Mutagenicity Test Kit Instruction Manual (positive control frequencies are at least 2 times of negative control counts (spontaneous frequency)). The results of negative and positive controls confirm the sensitivity and accuracy of the test system.
Please also refer to tables 1 to 3 in the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: Historical data of colonies per plate (negative control preparations without metabolic activation) 

Strain

MIN

MAX

MEAN

SD

N

TA98

13

54

29

16

5

TA100

62

246

112

78

5

TA1535

7

28

14

8

5

TA1537

3

8

5

2

4

WP2uvrA

10

17

14

3

4

Table 2: Historical data of colonies per plate (negative control preparations with metabolic activation)

Strain

MIN

MAX

MEAN

SD

N

TA98

21

65

38

19

5

TA100

63

232

108

70

5

TA1535

8

20

11

5

5

TA1537

3

6

5

1

4

WP2uvrA

10

20

15

5

4

Table 3: Historical data of colonies per plate (positive control preparations)

Strain

Chemical

MIN

MAX

N

TA98

2-Aminoanthracene

135

> 1000

5

Daunomycin

198

> 500

5

TA100

2-Aminoanthracene

326

> 500

5

Sodium Azide

274

> 500

5

TA1535

2-Aminoanthracene

67

> 300

5

Sodium Azide

83

> 300

5

TA1537

2-Aminoanthracene

107

210

4

ICR 191 Acridine

82

2010

4

WP2uvrA

2-Aminoanthracene

309

407

4

4-Nitroquinoline-1-oxide

839

1419

4

 

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.