Diarsenic trioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Studies described in the publication are acceptable for assessment.

Data source

Reference

Reference Type:

publication

Title:

Absence of arsenite mutagenicity in E. coli and Chinese hamster cells

Author:

Rossman, T.G. et al.

Year:

1980

Bibliographic source:

Environmental Mutagenesis 2: 371 - 379

Materials and methods

Principles of method if other than guideline:

An investigation of the mutagenicity of sodium arsenite in E. coli was performed. Spot tests, treat and plate protocols, and fluctuation test were conducted. Also, the potential of λ prophage induction was tested.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium dioxoarsenate

EC Number:

232-070-5

EC Name:

Sodium dioxoarsenate

Cas Number:

7784-46-5

IUPAC Name:

sodium dioxoarsenate(1-)

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

E. coli, other: WP2 (trpE), WP2s (trpE, uvrA), WP6 (trpE, polA1), WP10 (trpE, recA1), WP44s-NF (trpE, uvrA, tif-1/sfi-), WP44s-NF amp^r and WP2s (λ)

Metabolic activation:

not specified

Test concentrations with justification for top dose:

Spot test: 10 µL of 0.01M sodium arsenite (WP44s-NF (tif-1); 50 µL of 0.1 M sodium arsenite WP44s-NF, WP2, WP6, or WP10
Spot test: 50 and 100 µL of 0.1 M sodium arsenite
Treat and Plate Protocol: 25 mM sodium arsenite (WP2 & WP6)
Fluctuation test: 0.4, 1.0 and 2.0 mM arsenite (WP2)
Prophage induction: 1 and 10 mM arsenite (WP2s (λ))

Details on test system and experimental conditions:

MEDIA:
Cultures were grown in MST broth, consisting of Minimal Broth Davis (Difco) containing 0.2% glucose and 20 µg/mL tryptophan. Semi-enriched (SEM) plates contained Minimal Broth Davis (Difco), 0.2% glucose, and 5% nutrient broth (BBL), solidified with 1.5% agar. Nutrient plates contained nutrient broth solidified with 1.5% agar. Soft agar containing 6.5 mg Difco agar per liter of distilled water was ued to plate phage and bacteria.

BACTERIAL MUTAGENESIS
Plate assays for Trp+ revertants were performed by plating cells on SEM agar, according to the method of Witkin(1974)*. Plates were scored after three days of growth. Unless otherwise stated, the incubation temperature was 37°C.
Spot test for Trp+ revertants were performed by placing a sterile 12.7 mm diameter filter paper disc in the center of an SEM agar plate spread with approximately 2 X 10^7 bacteria. The test substance is added to the disc, and plates are scored for Trp+ revertants after three days of incubation at 37°C unless otherwise stated.
When toxic doses of arsenite are used, the bacteria can be exposed for only short periods of time and are then plated in the absence of arsenite (treat and plate protocol). these experiments were performed by treating a culture in exponential growth in MST broth with arsenite, removing samples at various times, centrifuging the cells, resuspending in the same volume of minimal A salts, and spreading 100 µL onto SEM agar plates.
Fluctuation tests for Trp+ revertants were carried out according to the method of Green et al. (1977)*.

INDUCTION OF λ PROPHAGE
The lysogen WP2s (λ) and indicator strain WP44s-NF-amp°r are grown in MST to midexponential phase. After treatment of the lysogen, infectious centers are assayed using a soft agar overlay on nutrient broth plates with or without 10 µg/mL ampicillin. Since the indicator strain WP44s-NF-amp^r carries the tif-1 mutation, lysogeny is not maintained well at 37°C and clear plaques result, allowing ease of scoring.

* References:
Witkin EM (1974): Thermal enhancement of ultraviolet mutability in a Tif-1 uvrA derivative of Escherichia coli B/r: Evidence that ultraviolet mutability in a Tif-1 uvrA derivative of function. Proc Natl Acad Sci 71: 1930-1934.
Green MHL, Rogers AM, Muriel WJ, Ward AC, McCalls DR (1977): Use of a simplified fluctuation test to detect and characterize mutagenesis by nitrofurans. Mutation Res 44: 139-143.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli, other: WP2, WP6, WP10 and WP44s-NF in spot test

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Test results 2

Species / strain:

E. coli, other: WP2s (λ) & WP44s-NF in spot test

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Test results 3

Species / strain:

E. coli, other: WP2 and WP6 in treat and plate protocol

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

This concentration of arsenite causes a complete inhibition of growth, and a decrease in viable count to 35% of the control value in one hour.

Test results 4

Species / strain:

E. coli, other: WP2 in fluctuation test

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Species / strain:

E. coli, other: WP2s (λ) in test on prophage induction

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Arsenite is not mutagenic to E. coli.