N,N-dibutylbutan-1-aminium bis[2-(hydroxy-kO)benzoato(2-)-kO]borate(1-)

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:

April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline (OECD 471) compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-dioxo-4H,4'H-2,2'-spirobi[[1,3,2]benzodioxaborinin]-2-uide; tributylazanium

EC Number:

700-906-2

Cas Number:

22450-96-0

Molecular formula:

C26H36BNO6

IUPAC Name:

4,4'-dioxo-4H,4'H-2,2'-spirobi[[1,3,2]benzodioxaborinin]-2-uide; tributylazanium

Constituent 2

Reference substance name:

SABoTBA

IUPAC Name:

SABoTBA

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): SABoTBA
- Purity: 98.77%
- Purity test date: 08 April 2014
- Lot/batch No.: 12342
- Expiration date of the lot/batch: Stable under recommended storage conditions
- Storage condition of test material: Room temperature in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

First test
Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 µg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both. In the absence of any toxic effects, the maximum concentration selected for use in the second test is the same as that used in the first. If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate is observed on the plates at the end of the incubation period, at least one precipitating concentration should be included in the second test.

Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen for the Salmonella strains was 1500 µg/plate, and the maximum concentration chosen for the E. coli strain was again 5000 µg/plate. Six concentrations were used.

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:

cytotoxicity

Remarks:

at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It was concluded that SABoTBA showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

A bacterial reverse mutation test was conducted using the procedure described in the current OECD Guideline 471.

This study was performed to investigate the potential of the test material to induce gene mutations. The test material, SABoTBA, was tested in four strains of Salmonella typhimurium and one strain of Escherichia coli. The test material was dissolved in DMSO and was tested at 5, 15, 50, 150, 500, 1500 and 5000 µg/plate using the poured plate method, with and without metabolic activation. Solvent controls and positive controls were included in the test. The following strains were used in the study Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA (pKM101).

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation (S9). Each concentration, including the controls, was tested in triplicate.

No toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in any of the strains used up to the highest investigated close in both experiments.

The plates incubated with the test article showed normal background growth up to 1500 µg/plate for Salmonella typhimurium and

5000 µg/plate with and without metabolic activation.

Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the strains used. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

It was concluded that the test material showed no evidence of mutagenic activity when tested in bacterial test systems.