Hexachloroacetone

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

The Mutagenic Properties of Hexachloroacetone In Shortterm Bacterial Mutagen Assay Systems

Author:

Howard Zochlinski, Howard Mower

Year:

1981

Bibliographic source:

Mutation Research, 89/2, 137 ff

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexachloroacetone

EC Number:

204-129-5

EC Name:

Hexachloroacetone

Cas Number:

116-16-5

Molecular formula:

C3Cl6O

IUPAC Name:

hexachloropropan-2-one

Test material form:

other: neat and DMSO solution

Details on test material:

Hexachloroacetone was obtained from Aldrich (lot H 530-8). The HCA was tested either directly (without solvent) or dissolved in dimethylsulfoxide
(DMSO) obtained from Sigma (lot 54 C-0084). A stock solution of HCA in DMSO (35 mg/ml) was tested with suitable dilution, either immediately after mixing or after storage at temperatures not in excess of 20 °C. In addition these solutions were tested after heating for 30 sec in a Bunsen flame
which causes a color change (from colorless to brown, then yellow). Storage at room temperature (~25°C) for longer than 45 min will also produce
this color change.

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:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

E. coli WP2

Species / strain 4

Species / strain / cell type:

E. coli, other: rec-assay

Test concentrations with justification for top dose:

0.3 mL neat and solution in DMSO

Vehicle / solvent:

no vehicle and DMSO solution

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Ames test:
The ability of HCA to cause the formation of revertant colonies in the Ames strains TA98, TA100, TA1535, TA1537, and TA1538 was measured by
the procedure described by Ames et al. Hexachloroacetone, either directly or in DMSO solution, was added to 3.0 ml of minimal histidine soft agar.
The total amount of solution added from the stock or from the various dilutions never exceeded 0.3 ml; appropriate controls consisted of addition of pure DMSO and routinely gave counts of spontaneous revertants which were similar to negative controls run in the absence of DMSO. Spontaneous revertants routinely observed were as follows: TA98, 28 ± 6; TA100, 126 ± 10; TA1535, 41 ± 3; TA1537, 12 ± 2; and TA1538, 25 ± 3. Positive
controls were run as suggested by De Serres and Shelby and were as follows: TA100, sodium azide 2 µg/plate, 900-1300 colonies; TA98,
2-nitrofluorene 50 µg/plate, 2800-3500; TA1535, sodium azide 1 µg/plate, 2200-3000; TA1537, 9 aminoacridine hydrochloride monohydrate
150 µg/plate, 6000-7000; TA15-38, 4-nitro-o-phenylenediamine 100 µg/plate, 2000-3000. After addition of the test solution to 3.0 ml of soft agar, 0.1 ml of a 10*9 culture of the text bacterial strain was added and the mixture immediately mixed on a Vortex mixer and plated onto base agar plates. The plates were then incubated in the dark for 48 h at 37°C and the number of revertants was counted, either with the aid of a hand-held tally counter or a Biotran II Automated Colony Counter. All experiments were run in triplicate and each data point is the average of the 3 experimental values.
These points were within 25% of the average or the experiment was repeated. The mutation ratio (MR) was calculated by dividing the number of
revertant colonies on the experimental plate by the number of colonies on the appropriate control plate. A MR of 2.0 or greater was considered
significant and indicative of a positive result.

E. coli IVP-2 assay
E. coli WP-2, Tryp(-), was obtained from Stanley Venitt. The cells were cultured according to the procedure of Green. The HCA, either directly or in
varying concentrations in DMSO, was added to 3. 0 ml minimal tryptophan soft agar at 45°C and 0.1 ml of the E. coli WP-2 culture was then added.
The mixture was immediately mixed on a Vortex mixer and poured over the hard agar plates prepared according to Ames. The plates were incubated for 48 h at 37°C and then counted for revertant colonies. All experiments were run in triplicate. Spontaneous revertants routinely numbered 18-30 colonies and positive controls with 1.0 µg/plate methyl methanesulfonate routinely produced 300-400 colonies/plate.
All experiments were run in both the Ames assay and the WP-2 assay at concentrations of test chemical which gave no evidence of toxicity to the
background lawn.

E. coli rec-assay:
E. coli JC5519 (rec-BC) and the DNA repair competent parent strain, E. coli AB1157, were obtained from John Clark, Department of Molecular Biology, University of California, Berkeley, CA. The test for mutagenicity was carried out according to the procedure of Ichinotsubo et al. In this assay the
HCA in DMSO solution was added to a 5 mm diameter well cut in a nutrient broth hard agar plate which had been seeded with 107-108 cells of either the DNA repair deficient strain (JC5519) or the DNA repair competent strain (AB1157). The plates were incubated overnight at 37°C and the zones of no growth surrounding the well on each plate were measured. Larger zones of killing on the JC5519 plate compared to the AB1157 plate indicate that the test chemical causes DNA damage and is likely to be mutagenic. The results were recorded as the net diameter of the zone of inhibition after
subtracting the diameter of the zone of inhibition observed on the AB1157 from that observed on JC5519. Ail experiments were run in triplicate and each data point is the average of 3 experimental values of the net zones of inhibition. A minimal net zone of inhibition of 2. 0 mm was considered
significant and indicative of mutagenic activity. Negative controls with DMSO gave no zones of inhibition and positive controls with 10µg/well of
MNNG routinely gave net zones of inhibition of 10-14 mm.
All bacteria strains used in this study were grown in nutrient broth media, 25 g/l. The cultures were harvested after 16-20 h growth and contained
10exp8-10exp9 cells/ml.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not determined

Additional information on results:

The maximum mutation ratios (max. MR) achieved were 5.1 for TA100 and 10.4 for TA98, both figures recorded at concentrations of 26.25 mg/plate of HCA. When tested in DMSO solution, hexachloroacetone gave a more complicated pattern of results. There were three distinct patterns of
response. One when the DMSO solution was kept at 20°C,the second when the solution was heated, and the third when the DMSO contained small
amounts of water. If the solution was kept at 20°C or less, it remained colorless and gave the response curve on TA98. The max. MR was 11.55,
occurring at 1.75 mg/plate. On TA100, HCA gave a response curve, for a similar DMSO solution. When kept at room temperature for 45 min or
longer or heated 30 sec in a bunsen flame, the solution of hexachloroacetone and DMSO turned color, forming a yellow solution. The color formation could be hastened at room temperature by placing a flame heated pipette in the HCA-DMSO mixture. These colored solutions showed an increase in the number of net revertants at the max. MR point of the colored solution for TA100 but not for TA98. Both strains showed a shift of the max. MR
point to lower concentrations of HCA. There was also an increase in the toxicity of the solution. Theentire dose-response curve became more
compressed. With a solution of hexachloroacetone dissolved in DMSO kept at 20°C, a max MR of 2.94 at 0.7mg/plate was achieved, while with direct application, a max. MR of 6.31 at 35 mg/plate was achieved, with no sign of the curve decreasing. A maximum zone of inhibition was observed at
10 mg HCA/well. Upon standing overnight in a loosely stoppered vessel, a stock solution of hexachloroacetone and DMSO formed crystals by
absorption of H2O from the air. Upon isolation, these crystals had a melting point of 44-45°C. The crystals were examined by NMR, IR, and mass
spectrometry, and all these observations suggested that the crystals were hexachloroacetone monohydrate. Hexachloroacetone monohydrate was
not mutagenic in DMSO/H2O mixtures in any of the short term tests used in this study when tested in amounts up to 30 mg/plate.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous Effects of temperature, solvent and chemical reactions with the solvents still unclear.

Hexachloroacetone reverts the Ames strains TA98 and TA100 but not the non-plasmid strains TA1537, TA1535 and TA1538.
Further experiments are necessary.

Executive summary:

Hexachloroacetone reverts the Ames strains TA98 and TA100 but not the non-plasmid strains TA1537, TA1535 and TA1538.

In the absence of solvent, the number of revertant colonies is 5 times the spontaneous reversion rate for TA100 and 10 times the spontaneous reversion rate for TA98 with 26 mg hexachloroacetone per plate. This effect is seen in the absence of rat liver microsomes. In dimethylsulfoxide (DMSO) solution a more complicated pattern is seen. In DMSO solution cooled between 18 and 20°C, the maximum number of revertants is similar to that found in the absence of DMSO, but only 1.75 mg hexachloroacetone per plate is needed. When DMSO solution of hexachloroacetone is warmed above 20°C, a yellow color develops and the solution becomes more toxic to the test bacteria. The maximum number of revertants is then produced at about 0.5 mg hexachloroacetone per plate. Hexachloroacetone is found to be active, without microsomal activation, in the E. coli WP-2 and E. coli rec-BC test systems. Hexachloroacetone readily reacts with water in DMSO solutions to form the non-mutagenic hexachloroacetone hydrate.

The testing of hexachloroacetone in the Ames Salmonella histidine reversion assay, the E. coli WP-2 assay and the E. coli rec-assay, all show that the compound is a genotoxic material. What is of particular interest is the effect of adding of solvent, DMSO, to the hexachloroacetone. DMSO has long been considered an inert solvent. The results herein indicate that the DMSO either reacts with the hexachloroacetone, or acts as a catalyst for some other reaction involving hexachloroacetone. The nature of the products formed in the heated solutions are unknown at this time. In addition to these effects, DMSO appears to be effective in causing the reaction of water with HCA to form the non-mutagenic hexachloroacetone hydrate.