p-benzoquinone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The Salmonella mutagenicity assay was carried out with a 20-min preincubation procedure under yellow lamps (Maron and Ames, 1983; Hakura et al., 1994). Dimethyl sulfoxide was used as the solvent of the BQs. Oxoid nutrient broth No. 2 was used for overnight culture. The $9 prepared from male Sprague-Dawley rat liver pretreated with phenobarbital and 5,6-benzoflavone, and the cofactors were purchased from Oriental Yeast Co. (Tokyo, Japan). To assess the effect of SOD and catalase on BQ-induced mutagenicity, 39 units and 26 units, respectively, were used. Two plates were used for each dose, and each experiment was conducted at least twice.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

p-benzoquinone

EC Number:

203-405-2

EC Name:

p-benzoquinone

Cas Number:

106-51-4

Molecular formula:

C6H4O2

IUPAC Name:

1,4-benzoquinone

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium, other: TA2637

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium, other: TA104

Metabolic activation:

without

Controls

Untreated negative controls:

yes

Details on test system and experimental conditions:

The Salmonella mutagenicity assay was carried out with a 20-min preincubation procedure under yellow lamps. Dimethyl sulfoxide was used as the solvent of the test material. Oxoid nutrient broth No. 2 was used for overnight culture. Two plates were used for each dose, and each experiment was conducted at least twice.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA104

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium, other: TA2637

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium, other: TA104

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium, other: TA2637

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Any other information on results incl. tables

p-BQ and 2,3-diCI-5,6-diCN-BQ were mutagenic for strain TA104, which is sensitive to oxidative mutagens suggesting that the mutagenicity of BQs for S. typhimurium is attributable to oxidative injury. To confirm this, we conducted an experiment to inhibit activated oxygen species scavengers. Thus, strain TA104 was exposed to the two most potent mutagenic BQs, p-BQ and 2,3-diCI-5,6-diCN-BQ, in the presence and absence of SOD and/or catalase in phosphate buffer. As shown in Fig. 3, catalase had a significant inhibitory effect on the mutagenicity of both BQs, indicating that activated oxygen species, at least H202, are involved in the mutagenicity of BQs for S. typhimurium TA104.

No correlation was, however, found between the mutagenicity of the BQs for TA104 strain and their one-electron reduction potentials E(Q/Q-) (mV). BQ derivatives possessing high one-electron reduction potentials are known to have poor ability to redox cycle between the quinones and the semiquinones, and to produce activated oxygen species, since electron transfer from the semiquinones to oxygen becomes electrochemically unfavorable (Powis and Appel, 1980; Powis et al., 1981; Hassan and Fridovich, 1979). Therefore, a probable explanation for the mutagenicity of p-BQ and 2,3-diC1-5,6-diCN-BQ for strain TA104 is that the semiquinone radicals of rapidly formed glutathione or amino acid conjugates of the BQs (Lau et al., 1988; Gant et al., 1986) or the corresponding hydroquinones are autooxidized to produce activated oxygen species. In any case, oxidative stress can be involved in BQ mutagenicity.

Five of the 12 BQs used were also mutagenic for strain TA2637, which is sensitive to frameshift mutagens. This indicates that the mutagenicity of BQs is attributable to BQ-DNA adducts that form with BQs having electrophilic electrophilic substituents. This adduct formation is supported by the observation that the mutagenicity of two chlorinated BQ derivatives was also detected with TA100, which is sensitive to basepair substitution mutagens. All of the BQs used in the present study, with the exception of duroquinone, possessed strong electrophilic properties or arylating ability (unpublished result). In fact, Phe-BQ (Horvath et al., 1992; Pathak and Roy, 1992), tert-butylbenzoquinone (Morimoto et al., 1991), and p-BQ (Levay et al., 1991; Zhang et al., 1993) have been reported to produce DNA adducts.

The mutagenicity of four of the seven mutagenic BQs was decreased by the presence of S9 mix, and the effect of S9 mix was complex, depending on the substituents of the BQs. The reason for this complexity of the effect of S9 mix on BQ-induced mutagenicity is probably the difference in the ratio of mutagenic activity to the cytotoxicity of each compound in the presence and absence of S9 mix.

One of the causes of the decrease in the mutagenic activity of BQs in the presence of S9 mix may be the detoxification process by which BQs are reduced to their corresponding hydrobenzoquinones via flavoprotein reduction enzymes present in S9 mix (Iyanagi and Yamazaki, 1970; Lind et al., 1982; Wefers and Sies, 1983). Another cause may be the prevention of oxidation by the stabilization of hydroquinones, which are produced via the reduction of corresponding BQs within the cells and/or the S9 mix medium without the ceils, by the reductase or reducing agents such as NADPH or NADH present in the S9 mix. In fact, we have found the mutagenicity of p-hydrobenzoquinone to be much weaker than that of p-benzoquinone (unpublished result).

Further investigations are, however, needed to better understand the mutagenicity of the quinones.

Applicant's summary and conclusion

Conclusions:

The Ames-Test for p-benzoquinone is ambiguous.

Executive summary:

p-Benzoquinone (p-BQ) showed the most potent mutagenic activity (17 induced revertants/nmol/plate for strain TA104 without S9 mix) among the BQs tested. TA104, which is sensitive to oxidative mutagens, was the most sensitive to the mutagenicity of the BQs of the five strains used, while the second most sensitive strain was TA2637, which detects bulky DNA adducts. Significant reductions in the mutagenicity of p-BQ, and 2,3-diC1-5,6-diCN-BQ without S9 mix were observed in the presence of catalase. These findings suggest that the mutagenicity of BQs for S. typhimurium is attributable to oxidative injury after BQ reduction and to DNA adducts that form with BQs that have electrophilic substituents.