Borate(1-), bis[ethanedioato(2-)-.kappa.O1,.kappa.O2]-, potassium (1:1), (T-4)-

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:

2004-10-05 to 2004-11-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant genetic toxicity study for lithium bis(oxalato)borate was used for read across and evaluation of genetic toxicity of potassium bis(oxalato)borate on the basis of structural similarity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

, Published 2000-06-08

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

, adopted 1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

244761-29-3

EC Number:

607-383-9

Cas Number:

244761-29-3

IUPAC Name:

244761-29-3

Constituent 2

Reference substance name:

Lithium bis(oxalato)borate

EC Number:

456-990-3

EC Name:

Lithium bis(oxalato)borate

IUPAC Name:

lithium bis(oxalato)borate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

SMILES: O=C1O[B-]2(OC1=O)OC(=O)C(=O)O2.[Li+]

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– -> trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

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:

S9-mix (Rat liver induced with Aroclor 1254)

Test concentrations with justification for top dose:

Combined range finding test/first mutation experiment:
TA1535, TA1537, TA98, TA100 und WP2uvrA without and with 5 % (v/v) S9-mix:
10, 33, 100, 333, 1000, 3330, 5000 µg/plate
Second mutation assay: without and with 10 % (v/v) S9-mix:
10, 33, 100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 100 without S9 mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2 uvrA without S9 mix

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For all tester strains with S9 mix

Details on test system and experimental conditions:

Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture ( 109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in Milli-Q water, and either 0.5 ml 89-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
The revertant colonies (histidine independent c.q. tryptophan independent) were counted manually if less than 40 colonies per plate were present. lf more than 40 colonies were present, these could be counted automatically with a Protosmodel 50000-colony counter.

Evaluation criteria:

Data evaluation and statistical procedures:

No formal hypothesis testing was done.
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagentic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance did not precipitate in the top agar. Precipitation of lithium bis(oxalato)borate on the plates was not observed at the start or at the end of the incubation period in all tester strains. All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related,
two-fold, increase in the number of revertants in two independently repeated experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Read across for evaluation of genetic toxicity of potassium bis(oxalato)borate from the Ames test study results of lithium bis(oxalato)borate is justified on the basis of structural similarity of the substances. The bis(oxalato)borate salt part [B(C2O4)2] is the same in both substances and only the cationic part of salts differ (lithium or potassium). Both lithium and potassium belong to alkali-metals.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Lithium bis(oxalato)borate is not mutagenic in the Salmonella Typhimurium reverse mutation assay and the Escherichia Coli reverse mutation assay.
Based on structural similarity, the result can be used for read across when evaluating genetic toxicity of potassium bis(oxalato)borate. The result can be used for classification purposes. For risk assessment, also potassium bis(oxalato)borate can be regarded as not mutagenic. See also attachment in section 13 (Analogue approach justification).

Executive summary:

Lithium bis(oxalato)borate was assayed for mutation in four strains of Salmonella typhimurium and one strain of Escherichia coli. according to OECD guideline 371 and EU method B.13/14. It did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies, in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Lithium bis(oxalato)borate is not mutagenic in the Salmonella Typhimurium reverse mutation assay and the Escherichia Coli reverse mutation assay. (NOTOX, 2004)

Based on structural similarity, the result can be used for read across when evaluating genetic toxicity of potassium bis(oxalato)borate. Consequently, also potassium bis(oxalato)borate can be regarded as not mutagenic.