Poly(polyalkylammonium) poly(µ-alkoxy)-µ6-oxido-polyhedropoly(chloridometal)ate polyalkanol solvate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the present test conditions the test substance was tested up to a cytotoxic concentration of 316 µg/plate caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation (LPT 2013).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-19 to 2013-01-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; GLP study without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

OPPTS changed its name to OCSPP,

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci resposible for histidine auxotropy

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- obtained from Trinova Biochem according to Dr. Bruce N. AMES,

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9; male rats

Test concentrations with justification for top dose:

Plate incorporation test: 1.0, 3.16, 10.0, 31.6, 100 and 316 µg per plate;
Preincubation test: 1.0, 3.16, 10.0, 31.6, 100 and 316 µg per plate

Vehicle / solvent:

50 mg of test item were completely dissolved in 10 mL aqua ad iniectabilia by using an ultrasonic bath (45 kHz) at 37°C for 60 minutes to a
concentration of 5 mg/mL. In the preliminary test the administration volume had to be increased from the generally employed 100 μL to
1000 μL per plate to achieve a concentration of 5000 μg/plate. In the main study an administration volume of 100 μL/plate was used for all
concentrations. The vehicle served as the negative control. Fresh preparations of the test item were used for the treatment in all experimental parts.

Untreated negative controls:

no

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

aqua ad iniectabilia

True negative controls:

no

Positive controls:

yes

Remarks:

for details see below

Positive control substance:

other: without metabolic activation: sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 2-nitroflurene in DMSO for TA 98, 9-amino-acridine in ethanol abs. for TA 1537, Mitomycin C in DMSO for TA 102

Remarks:

with metabolic activation: 2-aminoanthracene in DMSO for TA 100, TA 1535, Benzo(a)pyrene in DMSO for TA 98, TA 102, TA 1537

Details on test system and experimental conditions:

Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain
TA100. Ten concentrations ranging from 0.316 to 5000 µg test item/plate were tested. Cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at a concentration of 316 μg/plate.
Hence, 316 μg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction, the protein content of the S9 fraction was 33.1 mg/mL S9, cytochrome
P-450: 0.40 nmol/mg protein
ADMINISTRATION
- Dosing:
* Plate incorporation test: 1.0, 3.16, 10.0, 31.6, 100 and 316 µg per plate
* Preincubation test: 1.0, 3.16, 10.0, 31.6, 100 and 316 µg per plate
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
* sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 10 µg/plate
* 2-nitroflurene in DMSO for TA 98, 10 µg/plate
* 9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
* Mitomycin C in DMSO for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100 and TA 1535, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, TA 102 and 1537, 10 µg/plate
- negative control: the vehicle aqua ad iniectabilia was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: approximately 10E8 viable cells in the late exponential or early stationary phase

DETERMINATION OF CYTOTOXICITY
- Method: In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation cytotoxicity (scarce
background lawn and reduction of the number of revertants) was noted at the top concentration of 316 μg/plate.

Evaluation criteria:

The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a
positive response if
- at one or more concentrations the number of revertants is reproducibly increased in at least one strain with or without metabolic activation. A
2-fold increase in comparison to the solvent control is regarded as being relevant for a positive response in the strains TA98, TA100 and TA102. For the strains TA1535 and TA1537 a 3-fold increase represents a biological relevant effect. The Mann and Whitney test (p ≤ 0.05, see section 6,
reference 3.) may be used to determine statistical significance.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation
coefficient (section 6, reference 3.) may be applied.
Biological relevance of the results should be considered first.
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.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Statistics:

According to the OECD Guideline 471, a statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium 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:

was noted at the top concentration of 316 μg/plate, in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

CYTOTOXICITY EFFECTS:
- plate incorporation test without and with metabolic activation: was noted at the top concentration of 316 μg/plate
- preincubation test without metabolic activation: was noted at the top concentration of 316 μg/plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

see attchached document

Conclusions:

Interpretation of results (migrated information):
negative

Under the present test conditions the test item tested up to a cytotoxic concentration of 316 µg/plate caused no mutagenic effect in the Salmonella
typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The purpose of this study was to evaluate the test substance for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

The test item was examined in the 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

Test item was completely dissolved in aqua ad iniectabilia.The vehicle served as the negative control.

Preliminary test

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg test item/plate were tested.Cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted startingat a concentration of 316 µg/plate.

Hence, 316 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 1.0 to 316 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation cytotoxicity (scarce background lawn and reduction of the number of revertants) was notedat the top concentration of 316 µg/plate.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a cytotoxic concentration of 316 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The 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.

 

In conclusion, under the present test conditions the test item tested up to a cytotoxic concentration of 316 µg/plate caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Under the present test conditions Indate (2-), hexachlorododeca-µ-methoxy-µ6-oxohexa-, hydrogen, compd. with N-methylmethanamine (1:2:2) caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Justification for selection of genetic toxicity endpoint
Annex VII requires an in vitro gene mutation assay in bacteria. Therefore the Salmonella reverse mutation assay has been chosen.

Justification for classification or non-classification

According to the EC Regulation 1272/2008 and subsequent regulations on classification, labelling and packaging of substances and mixtures, the test item is not genotoxic and has not to be classified.