Diquat dibromide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Dec 1985 to 20 Dec 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diquat dibromide

EC Number:

201-579-4

EC Name:

Diquat dibromide

Cas Number:

85-00-7

Molecular formula:

C12H12N2.2Br

IUPAC Name:

1,1'-ethylene 2,2'-bipyridyldiylium dibromide

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Method

Target gene:

his- (S. typhimurium), trp- (E. coli)

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:

Type and composition of metabolic activation system:
- Source of S9: induced rat liver S9 from six male rats dosed with Aroclor 1254.
- method of preparation of S9 mix: After sacrifice, each animal was dissected using sterile instruments to expose the liver. The liver was removed in lobes and placed in a beaker of ice-cold buffer (see below). After all the livers were removed, the pieces were washed in ice-cold buffer, then transferred to a pre-weighed beaker containing a measured volume of (ice-cold) buffer. The beaker was then reweighed and the liver weight calculated. The remaining volume of buffer to make a 25 % w/v homogenate was then measured out and stored on ice until required. Pieces of liver were then added, together with buffer from the beaker, to the pre-sterilised tube of a homogeniser, and homogenised with eight passes at 1070 rpm. The homogenate was poured into a sterile conical flask, and kept on ice until all the livers had been homongenised. The rest of the measured buffer was then added to the flask and the contents mixed by swirling. Equal volumes were then added to pairs of centrifuge tubes kept on ice. After being tightly capped the tubes were placed in a pre-cooled 8 x 50 mL rotor, and centrifuged at 9000 x g for 10 minutes in a refrigerated centrifuge with the temperature regulated at 4°C. The timing sequence was started once the rotor reached full speed. The resultant supernatants (S9 fraction) were combined and divided into 13 mL aliquots, rapidly frozen and stored at -70 °C. A 0.5 mL sample of the S9 fraction was added in duplicate to nutrient agar plates. The plates were incubated for 48 hours at 37 °C and assessed for contamination. When required, an aliquot of S9 fraction was thawed at room temperature until ice was no longer visible. It was then stored on ice until used.
- S9 Buffer: This was prepared as follows components with final concentrations: Sucrose 250 mM, Tris base 50 mM, EDTA tetrasodium salt (dihydrate) 1.0 mM. These were dissolved in approximately 4800 mL de-ionised H2O, the pH adjusted to 7.5 and the volume made up to 5000 mL. The solution was then stored at 4°C until required. Aliquots were sterilised by filtration through a 0.45 μm filter as needed.
- Co-factor Solution: The co-factor solution was made up from the following with their final concentration: 100 mM Na2HPO4, 33 mM KCl, 5 mM Glucose-6-Phosphate, 4 mM NADP (Na salt), 4 mM NADPH, 8 mM MgCl2. This was dissolved in 180 mL de-ionised H2O, the pH adjusted to 7.4 and the volume made up to 200 mL. The solution was then divided into 10 mL and 20 mL aliquots and stored at -20 °C as required. When required. sufficient aliquots were thawed by standing at room temperature until no ice was visible, sterilised by filtration through an 0.45 µm filter then stored on ice until used.

Test concentrations with justification for top dose:

All concentrations given as µg test substance cation
- Preliminary cytotoxicity assay: 10, 50, 100, 500, 1000, 5000 µg/plate
- Main assay (without metabolic activation): 0.1, 0.5, 1, 5, 10, 50 µg/plate, plus 0.01 and/or 0.05 µg/plate in repeat experiments
- Main assay (with metabolic activation): 0.5, 1, 5, 10, 50, 100 µg/plate, plus 0.01 and/or 0.05 µg/plate in repeat experiments

Vehicle / solvent:

- Solvent used: dionised water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine (4-NOPD); 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2); N-Methyl-N´-nitro-N-nitrosoguanidine (MNNG)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

EXPERIMENTAL PERFORMANCE
The presence of the uvrB deletion (Salmonella) and the uvrA mutation (E.coli) were confirmed by testing the sensitivity of each culture to mitomycin C (10 µL of a 10 µg/mL solution) in the same manner as sensitivity to crystal violet was tested. Damage to DNA caused by mitomycin C is repaired in normal bacteria by the uvr excision repair pathway, and is thus toxic to strains deficient at either the uvrA or uvrB loci. When fresh frozen stocks were prepared (from the frozen permanent strains), the Salmonella strains were tested for histidine requirement and for reversion properties using diagnostic mutagens as described by Ames et al (1975) and Maron and Ames (1983), except that the mutagens were incorporated in the top agar layer as in a normal test (see below) rather than spot tested as stated by Ames. All experiments were conducted using frozen stock cultures prepared as above in October 1985 (S.typhimurium strains), and in November 1980 (E.Coli)

Post-mitrochondrial supernatant (S9-mix)
The variant of the routine Ames assay used for this work was conducted with and without S9-mix incorporated in the top-agar. The S9-mix was prepared as follows in expressed volumes per 30 mL S9-mix:
- 3 mL S9 fraction
- 7 mL Sucrose-Tris-EDTA Buffer
- 20 mL Co-factor solution
In tests without metabolic activation, the S9-mix was replaced by an equivalent volume of phosphate buffer. Both the S9-mix and the phosphate buffer were kept on ice until used. Unused S9-mix was discarded at the end of the day.

Agar Plates
9 cm diameter vented Petri-dishes pre-poured with Vogel Bonner minimal medium and containing 1.5 % w/v agar and 2 % w/v glucose. The plates were stored at ambient temperature until needed to dry them and ensure sterility.

TYPE OF BACTERIAL ASSAY
Standard plate test (with and without S9). Toxicity was seen in the first main experiment and so lower dose levels were assessed in Experiment 2. Due to contamination seen in this experiment, repeat data were generated for strain TA98 in a 3rd experiment.

PROTOCOL
Bacterial cultures were prepared from frozen stocks by incubating overnight at 37°C in a shaking incubator. The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each dose level, solvent and positive controls;
- 500 μL S9 mix or phosphate buffer;
- 2 mL Overlay agar containing 0.5 μM histidine / 0.5 μM biotin or 1.03 μM tryptophan, as appropriate.
The mixture was poured onto the surface of a prepared plate and allowed to gel. After the agar was set the plates were incubated upside down for 64 - 68 hours at 37 °C in the dark. For each strain and dose level, including the positive controls three plates were used. For negative controls 2 or 5 plates were used. Following incubation all plates were counted using an automated colony counter with the discriminators adjusted to a standard setting which only permits the counting of mutant colonies.

Evaluation criteria:

A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:
- a significant, dose-related increase in the mean number of revertants is observed;
- a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) is observed at one or more concentrations

A negative result in a (valid) individual experiment is achieved when:
- there is no significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and
- in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.

For a positive response in an individual experiment to be considered indicative of an unequivocal positive, i.e. mutagenic, result for that strain/S9 combination, then the observed effect(s) must be consistently reproducible.

Statistics:

One-tailed Student’s t-test. The corresponding probability for each dose level was determined from a t-table using the appropriate degrees of freedom.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY ASSAY
The test substance induced significant toxicity in strain TA100 (both with and without S9) at doses of 50 μg test substance cation/plate or greater. At 100 μg/plate, the observed toxicity was more extreme in the absence of S9 than in the presence of S9. The doses ranges selected for the first full experiment were therefore 100 – 0.5 μg test substance cation/plate (+S9) and 50 – 0.1 μg test substance cation/plate (-S9).

MUTAGENICITY ASSAY
In the first full experiment, the test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used, either in the presence or absence of S9-mix, at any sub-toxic dose. In view of the range of toxic effects seen in this experiment, the repeat experiment was conducted using a range of lower dose levels in the presence or absence of S9-mix (5.0 – 0.01 μg/plate (-S9) and 10 – 0.05 μg/plate(+S9). Contamination was seen with strain TA98 in the second experiment and so repeat data were generated for this strain in a third experiment. No significant increases in revertant colony numbers were observed in any strain, with significant toxicity being observed in (at least) the top dose tested in each case.

The positive controls for each experiment induced the expected responses, indicating the strains were responding satisfactorily in each case. Revertant colony numbers for the solvent control plates were within acceptable ranges.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this assay, the test substance gave an unequivocal negative, i.e. non-mutagenic response, in both the presence and absence of an auxiliary metabolising system (S9) in all five Salmonella typhimurium tester strains used (TA1535, TA1537, TA1538, TA98 and TA100), and also in one strain of Escherichia coli (WP2 uvrA pKM101).

Executive summary:

In a reverse gene mutation assay in bacteria, according to OECD TG 471 and in compliance with GLP, strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium and WP2uvrA pkM101 of E. coli were exposed to the test substance at concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50, or 100 µg test substance cation/plate in the presence of mammalian metabolic activation (plate co-incubation) and at concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, or 50 µg test substance cation/plate in the absence of mammalian metabolic activation, S9 (plate co-incubation). The test substance was tested up to cytotoxic concentrations (up to 5000 µg/plate) in an initial dose-ranging study.

In two separate experiments, the compound did not induce any significant increase in the observed numbers of revertant colonies in any of the tester strains used, neither in the presence or absence of an auxiliary metabolising system (S9). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In conclusion, the test substance gave an unequivocal negative, i.e. non-mutagenic, response in this assay.