Complexation products of bismuth(3+) neodecanoate with propane-1,2-diol, propoxylated and 2,2',2'',2'''-ethylenedinitrilotetraethanol

Genetic toxicity: in vitro

Administrative data

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:

2017-12-13 to 2018-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

signed on 2 November 2016

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Complexation products of bismuth(3+) neodecanoate with propane-1,2-diol, propoxylated and 2,2',2'',2'''-ethylenedinitrilotetraethanol

EC Number:

948-264-4

IUPAC Name:

Complexation products of bismuth(3+) neodecanoate with propane-1,2-diol, propoxylated and 2,2',2'',2'''-ethylenedinitrilotetraethanol

Test material form:

liquid

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation by a microsomal fraction of rat liver (S9-mix 10%v/v)

Test concentrations with justification for top dose:

- Concentrations for assay n°1: 50, 150, 500, 1500 and 5000 µg/plate
- Concentrations for assay n°2: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Solutions were prepared in DMSO.

Controls

Controls 1

Untreated negative controls:

yes

True negative controls:

yes

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Remarks:

Test item was completely soluble in ethanol at all concentrations tested.

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Stock solution was formulated in DMSO.

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

NaCl 0.15 M

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Stock solution was formulated in NaCl 0.15 M.

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Stock solution was formulated in DMSO.

Controls 6

Negative solvent / vehicle controls:

yes

Remarks:

NaCl 0.15 M.

Positive controls:

yes

Positive control substance:

other: cis-Platinium (II) Diammine Dichloride: for E. Coli without S9

Remarks:

Stock solution was formulated in NaCl 0.15M.

Controls 7

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-anthramine: for TA1535, TA1537, TA98 and TA100 with S9

Remarks:

Stock solution was formulated in DMSO.

Controls 8

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Stock solution was formulated in acetone.

Details on test system and experimental conditions:

PREPARATION OF STOCK SOLUTION
-Test item is completely soluble in DMSO at all concentrations tested.
- Stock solution for each assay was prepared at 100 mg/mL in DMSO.
- Test item is tested at 5000, 1500, 500, 150 and 50 µg/plate.
- 50µL volume additions of test item solution were used for all treatments.

BACTERIA
- Strains of Salmonella typhimurium and Eschericia coli are purchased from Moltox. They are maintained in LEMI laboratory.
- The genotype of bacterial strains was checked.

EXPERIMENTS
- Triplicate plates test material.
- Negative, positive and vehicle controls were included in triplicate with and without metabolic activation.
- S9 fraction, microsome fraction, prepared from Sprague Dawley rat liver homogenate, was provided by Moltox USA.
- For experiments without metabolic activiation: 0.1 mL bacterial culture, 50µL of test article solution or control and 0.5 mL of buffer solution were added to 2 mL overlay agar at 45°C containing 10%v/v of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains and 5%v/v of nutrient broth n°2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/L for Escherichia coli strain. Plates are incubated at 37°C over 48-72 hour period.The pre-incubation phase is performed for the second assay if the first assay is negative.
- For experiments with metabolic activation: protocol similar to that described above, except that 500 µL of S9-mix fraction is quickly added before pouring the mixture onto the plates. Since the first assay was negative, the pre-incubation test was performed for the second assay.

COLONY COUNTING
- After a 48-72 hour incubation period at 37°C, revertant colonies are counted in each plate.

Evaluation criteria:

Ensure that the criteria of validity of the study are well respected namely:
- the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75%,
- the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory,
- negative and positive values should not show significant difference with the historical values of the laboratory (+/- 2 standard deviations).
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and E. coli WP2 (uvrA) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependant relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA98, TA100 and E. coli WP2 (uvrA) (pKM 101) and three fold for TA1535 and TA1537.
All results must be confirmed in an independant experiment.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for positive controls (with and without metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (5000, 1500, 500, 150 and 50 µg/plate) without and with metabolic activaiton in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2(uvrA)(pKM101).
We can observe for all strains tested a significant decrease in the number of revertant colonies in the presence of highest dose tested 5000 µg/plate.
Results are confirmed in an independant experiment.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Doses (5000, 1500, 500, 150 and 50 µg/plate) performed from solutions of the test item do not induce any mutagenic change in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Eschirichia coli WP2(urA-)(pKM101) without, or with metabolic activation, according to the OECD guidelines n°471.