Hexachloroacetone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Unsuitable test system

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Principles of method if other than guideline:

only two strains (TA 98 and TA 100) have been used.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

without

Positive control substance:

2-nitrofluorene

sodium azide

Details on test system and experimental conditions:

The experiments described here were performed on a sample of chlorination-stage effluent from a bleached kraft pulp mill in the interior of British
Columbia. This sample was subjected to a number of fractionation steps as described below and the mutagenic activity of each fraction assayed
using Salmonella typhimurium histidine-dependent strain TA100 without the addition of in vitro metabolic activation. Pure compounds identified in
this study were bioassayed subsequently with both strains TA98 and TA100. Characteristics and procedures for these strains, which previously gave the best responses to chlorination-stage effluents without metabolic activation, have been described.
The sample of effluent exhibited typical high mutagenicity predicted for chlorination-stage effluent and was toxic above 1.0 ml per plate. After
passage through an XAD-2 resin column most of the mutagenicity detected by strain TA100 was removed. Elution of the resin with ether followed by methanol and evaporation of the solvents resulted in a highly mutagenic ether eluate and a comparatively non-mutagenic methanol eluate.
The ether eluate, containing most of the mutagenicity detectable in strain TA100, was fractioned by adsorption chromatography on a silica-gel
column. Elution of this column with hexane : ether (1 :1) gave a fraction with relatively high mutagenicity in a weight basis.
The identity of this compound was suggested by strong peaks in the mass spectrum at m/e 83 and 85. Confirmation was obtained by comparison of the retention time of an authentic sample on a SP 2100 quartz capillary column and by the characteristic reaction with diazomethane which resulted
in a shift to a longer retention time.
To determine the relationship between the recovery of alkali-labile components and pH, samples of the original effluent were adjusted to various pH values. After 1h NaCl was added to 10% content and the samples extracted with ether, the extracts were concentrated and analyzed by capillary gas
chromatography on a SP 2100 column. Hexachloro-acetone was identified in extracts of the effluent at pH 2 by mixed injections with authentic
samples. Hexachloro-acetone was not observed in gas chromatograms of ether extract from effluent adjusted to pH 7 however tetrachloroacetone was still present.
The acetone produced strong dose-related mutagenic responses in Salmonella strains TA98 and TA100 without S9 addition when tested as pure
compounds at concentrations of less than 0.5 µl per plate. It is interesting to note that the degree of chlorine substitution appears to influence the mutagenic potency of chloroacetones, since the dose at which the maximum number of revertants/plate are observed increases as the number of
chlorine atoms increases.
The rapid disappearance of these chloroacetones from alkaline solutions is in agreement with the reported alkali-lability of the mutagenicity of chlorination stage effluent. This feature plus their strong direct-acting mutagenic effect suggests that these compounds are a major component of the
mutagenic activity of chlorinated-stage effluent. However, caution should be exercised in assuming that the mutagenic activity of an effluent mixture can be determined from the simple addition of the activities of the individual components comprising that mixture. Thus, the amount of chloro-
acetones present cannot actually be determined by comparing their mutagenic activities with that of the effluent.

Results of the experiment to determine the stability of chloroacetones in alkali indicated they are very labile and could best be recovered by extraction at low pH. An approximation of the concentrations of hexachloroacetone was obtained by extracting 2 samples of chlorination-stage effluent and measuring the quantities by gas chromatography. Methyl heptadecanoate was used as an internal standard andconcentrations were calculated on the basis of peak areas corrected for response factors but not for recovery from aqueous acid solution.

Hexachloroacetone appears to be more labile.

Although chloroacetones have been identified in samples of chlorination-stage effluent, their presence in combined whole-mill effluent after it has undergone biotreatment has not yet been determined, and the extent to which chloroacetones reach the environment outside the mill is unknown.

Conclusions:

Interpretation of results (migrated information):
positive

The hexachloroacetone produced strong dose-related mutagenic responses in Salmonella strains TA98 and TA100 without S9 addition when tested as pure compounds at concentrations of less than 0.5 µl per plate. It is interesting to note that the degree of chlorine substitution appears to
influence the mutagenic potency of chloroacetones, since the dose at which the maximum number of revertants/plate are observed increases as the number of chlorine atoms increases.
The rapid disappearance of these chloroacetones from alkaline solutions is in agreement with the reported alkali-lability of the mutagenicity of
chlorination stage effluent. This feature plus their strong direct-acting mutagenic effect suggests that these compounds are a major component of
the mutagenic activity of chlorinated-stage effluent. However, caution should be exercised in assuming that the mutagenic activity of an effluent
mixture can be determined from the simple addition of the activities of the individual components comprising that mixture. Thus, the amount of
chloroacetones present cannot actually be determined by comparing their mutagenic activities with that of the effluent.

Executive summary:

Certain effluents from pulp mills have been shown in the Salmonella/mammalian- microsome test and using a battery of microbial and mammalian in vitro assays to possess mutagenic activity. In these studies, first-chlorination-stage effluent from both bleached kraft and sulfite mills consistently showed high mutagenic activity where as, most other effluents and process streams showed no or reduced mutagenicity. These studies also revealed that, mutagenicity found in effluents could be detected without the addition of an in vitro metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)