3,4,5-trihydroxybenzoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The weight of evidence approach is justified as determined from the following 7 pieces of information (independent sources).

a) Chen & Chung (2000):

Gallic acid is not mutagenic in the Ames Salmonella mutagenicity assay system using TA98 and TA100. Gallic acid is not antimutagenic against direct mutagens such as 2-nitrofluorene, 2-nitro-p-phenylenediamine, 3-nitro-o-phenylenedia-mine, 4-nitro-o-phenylenediamine, 4,4'-dinitro-2-biphenylamine, 1-nitropyrene and 1,3-dinitropyrene using TA98.

b) Martinez et al. (2000):

Bacterial reversion assay called the WP2 Mutoxitest (based on strain IC203, deficient in OxyR, and its oxyRq parent WP2 uvrA/pKM101 (denoted IC188)) has been used in the evaluation of gallic acid (among 80 chemicals) for oxidative mutagenicity. Gallic acid caused oxidative mutagenesis, being positive only in IC203 and sensitive to inhibition by S9.

c) Sevgi et al. (2015) have investigated the antioxidant and DNA damage protection potentials of gallic acid using four different test systems named asβ-carotene bleaching, DPPH free radical scavenging, reducing power and chelating effect. Antioxidative activities of the extracts were compared with those of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) used as positive controls. This study compares the potential of gallic acid as antioxidant and DNA damage protector among other phenolic acids and in comparison with positive controls.

d) Using the cytokinesis-block micronucleus assay in WIL2-NS cells, Sugisawa et al (2004) investigated the effects of six tea constituents, including gallic acid (GA), on chromosomal damage in two ways; induction by each component on its own and prevention against treatment of reactive oxygen species (ROS). None of the tea constituents induced chromosomal damage at <10µM. On the other hand, GA prevented H2O2-induced chromosomal damage in a dose-dependent manner with a significant effect detected at 1µM. Chromosomal damage induced by tert-butylhydroperoxide was not prevented by GA even at 10µM. These results suggest that physiological concentration of tea constituents are not genotoxic but rather anti-genotoxic against ROS, although their preventive effects are slightly different depending on their chemical structure.

e) Yonezawa et al (2001) have applied the Mut-Test based on Escherichia coli tester to demonstrate that gallic acid is negative as antimutagen.

f) Birosova et al. (2005) have used Salmonella typhimurium tester strain TA 100 in the plate-incorporation test to examine the antimutagenic potential of different compounds including gallic acid, on 3-(5-nitro-2-furyl)acrylic acid (5NFAA) and sodium azide mutagenicity. All tested compounds possess antimutagenic activity with gallic acid identified with the highest antimutagenic potency of tested compounds.

g) Hour et al (1999) have tested the antimutagenic properties of various extracts and test items including gallic acid by the Ames test (TA100, TA98 and TA97). Gallic acid strongly inhibited the mutagenicity of 9AA, and moderately inhibited the mutagenicity of MNNG and folpet. Gallic acid perhaps could act as nucleophiles to scavenge the electrophilic mutagens.

h) Gichener et al (1987) have investigated the antimutagenic potential of gallic acid: the frequency of his + revertants induced by N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the strain TA100 of Salmonella typhimurium was decreased by gallic and tannic acid. In weak buffer solutions, the inhibition effects of gallic acid towards MNU and MNNG mutagenicity was caused primarily by a decrease of pH in the incubation mixtures. At adjusted pH (pH 5.0 and 6.5), the antimutagenic effects are largely the result of an interaction between MNU or MNNG with phenolic acids outside the cells.

i) Sloczynska et al (2014) have reviewed the antimutagenic properties of different compounds and their possible mode of actions. Hypothesis is that gallic acid could perhaps act as a nucleophile to scavenge the electrophilic mutagens. Moreover, it was implied that gallic acid can bind or insert into the outer membrane transporters and lead to the blockage of a mutagen that was transferred into the cytosol.

 

We claim the reliability, relevance, adequacy and quality of the weight of evidence. Each study alone is of low reliability (purity not detailed, poor description of protocol). Whereas the 5 combinations of strains recommended by OECD 471 have not been tested, the data package shows an absence of mutagenic effect of gallic acid and, in some experiment, an antimutagenic potential using in vitro testing. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the present study, the plate-incorporation test of the Salmonella mutagenicity assay was used to examine the effect of selected phenolic acids against 5NFAA and sodium azide mutagenicity.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 100

Test concentrations with justification for top dose:

30, 60, 120, 250, 500 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 3-(5-nitro-2-furyl)acrylic acid (5NFAA)

Details on test system and experimental conditions:

The inhibitory effect of phenolic acids on mutation induction by several positive mutagens was investigated with Salmonella typhimurium TA100 using pre-incubation method. 0.1 ml of the positive mutagen, 0.1ml of phenolic acid and 0.1 ml of bacterial culture (cultivation for 16 h at 37 °C, approximate cell density 2–5 × 108 cells/ml) were mixed and preincubated at 37 °C for 30 min. Soft agar (2 ml) was added and the mixture was poured onto minimal agar plates. After 48 h of incubation at 37 °C the number of revertants was counted. The results from the antimutagenicity studies represent the mean of three separate experiments, each run in triplicate, and they were statistically evaluated using the Student’s t-test. Antimutagenicity was expressed as percentage of mutagenicity inhibition following the formula: % mutagenicity = 100–[(X1/X2) × 100] where X1 = number of revertants per plate in the presence of mutagen and antimutagen, X2 = number of revertants per plate in the absence of antimutagen.

Statistics:

Results were statistically evaluated using the Student’s t-test.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not specified

Genotoxicity:

other: Inhibition of the mutagenicity

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The positive control of mutagen in each case was considered as 100 % mutagenicity. Gallic acid was the only phenolic acid successful in inhibiting the mutagenicity of 5NFAA by more then 50 % at the concentration of 500 μg/plate. All tested compounds (with the exception of cichoric acid) decreased the number of revertants induced by sodium azide by about 20–35 %. Similarly as in the case of 5NFAA, the effect of sodium azide was significantly reduced by gallic acid and within the concentration tested a marked dose-dependence was found. At the highest used concentration this phenolic acid inhibits mutagenic activity of sodium azide by 82 %.

Conclusions:

In the present study, the Salmonella typhimurium tester strain TA 100 was used in the plate-incorporation test to examine the antimutagenic potential of caffeic, ferulic and cichoric acids extracted from plant species of genera Echinacea (L) Moench, as well as of another phenolic acids, on 3-(5-nitro-2-furyl)acrylic acid (5NFAA) and sodium azide mutagenicity. All tested compounds possess antimutagenic activity. In the case of 5NFAA, the antimutagenic potency of tested compounds was in the order of gallic acid > ferulic acid > caffeic acid > syringic acid > vanillic acid.
The mutagenic effect of sodium azide was inhibited by tested phenolic acids by about 20–35 %. The most effective compound, gallic acid inhibits this effect by 82 % in the concentration of 500 μg/plate. The only exception from favourable properties of tested phenolic acids is cichoric acid, which in the contrary significantly increased the mutagenic effect of 5NFAA.

Executive summary:

In the present study, the Salmonella typhimurium tester strain TA 100 was used in the plate-incorporation test to examine the antimutagenic potential of caffeic, ferulic and cichoric acids extracted from plant species of genera Echinacea (L) Moench, as well as of another phenolic acids, on 3-(5-nitro-2-furyl)acrylic acid (5NFAA) and sodium azide mutagenicity. All tested compounds possess antimutagenic activity. In the case of 5NFAA, the antimutagenic potency of tested compounds was in the order of gallic acid > ferulic acid > caffeic acid > syringic acid > vanillic acid.

The mutagenic effect of sodium azide was inhibited by tested phenolic acids by about 20–35 %. The most effective compound, gallic acid inhibits this effect by 82 % in the concentration of 500 ?g/plate. The only exception from favourable properties of tested phenolic acids is cichoric acid, which in the contrary significantly increased the mutagenic effect of 5NFAA.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mutagenic activity of gallic acid has been tested using Ames Salmonella tester strains TA98 and TA100.
Antimutagenic activity of gallic acid against a number of direct mutagens including 2-nitro¯uorene (2-NF), 4,4'-dinitro-2-biphenylamine, 1-nitropyrene, 1,3-dinitropyrene, 2-nitro-p-phenylenediamine, 3-nitro-o-phenylene-diamine, 4-nitro-o-phenylenediamine has also been tested.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Gallic acid (149-91-7) was purchased from Sigma (St Louis, MO, USA). All solutions were freshly prepared by dissolving the chemicals in DMSO or in phosphate buffer and were kept in the dark.

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with

Metabolic activation system:

S9 mix (Aroclor 1254-induced, Sprague±Dawley male rat liver in 0.154 M KCl solution). Before use, the S9 mix was ®ltered through a 0.45 mm Nalgene disposable ®lter (Nalge Co., Rochester, NY, USA).

Test concentrations with justification for top dose:

375, 750, 1500 and 3000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

no

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminofluorene

Details on test system and experimental conditions:

Mutagenicity tests were performed using standard preincubation procedures and in the presence or absence of the liver S9 mix. All operations were conducted under yellow light to avoid photo-oxidation of the compounds.
The antimutagenic tests were performed similar to the standard plate incorporation and preincubation procedures of Maron and Ames (1983), except the antimutagens were also added and preincubated for 30 min before plating.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No tannic acid or its related compounds was mutagenic towards Salmonella tester strains TA98 and TA100 in the absence or presence of S9 mix The antimutagenicity testing of these compounds against nitro-group containing mutagens including 4,4'-dinitro-2-biphenylamine, 1-nitropyrene, 1,3-dinitropyrene, 2-nitrofluorene, 2-nitro-p-phenylenediamine, 3-nitro-o-phenylenediamine and 4-nitro-o-phenylenediamine to TA98 in the plate incorporation assay in the absence of metabolic activation showed that they did not affect the metabolic activity of these mutagens. However, when these compounds were tested against those mutagens that required metabolic activation, namely benzidine, 4-aminobi-phenyl, 3,3'-4,4'-tetraaminobiphenyl, and N,N-N',N'-tetramethylbenzidine, different results were obtained. The number of histidine revertants was signi®cantly lower in the presence of tannic acid at its non-growth inhibitory concentrations (<0.2 mmol/plate) than without the tannic acid. Tannic acid at higher concentration was inhibitory to the growth of the tester strain. Propylgallate, ellagic acid and gallic acid showed no antimutagenic activities against all mutagens tested. In this paper, we tested the anti-mutagenic activity of tannic acid at concentrations that were non-growth inhibitory to the tester strains. Gallic acid, ellagic acid and propyl gallate were not antimutagenic at these low concentrations. The possibility should not be excluded that anti-
mutagenic activity could be found at higher concentrations.

Mutagenicity of tannic acid and related compounds to Salmonella typhimurium TA98 and TA100

		Revertant colonies per plate (mean ± SD)
		TA98		TA100
Compound	Dose (per plate)	-S9	+S9	-S9	+S9
DMSO	100 µl	20 ± 3	20 ± 4	105 ± 28	103 ± 1
2-Nitrofluorene	4 µg	349 ± 20	Not tested	-
MNNG	1 µg	-	-	2112 ± 142
2-Aminofluorene	10 µg	-	4625 ± 30	-	2110 ± 135
Gallic acid (mg)	375	18 ± 4	26 ± 2	91 ± 5	78 ± 7
	750	11 ± 3	16 ± 7	126 ± 8	103 ± 7
	1500	12 ± 1	19 ± 4	94 ± 6	104 ± 14
	3000	16 ± 5	15 ± 9	86 ± 12	80 ±2

 

Effect of tannic acid and related compounds on the mutagenicity of nitro group-containing compounds in Salmonella typhimurium TA98

 

 

	Number of His+ revertants induced by mutagens/plate
Direct mutagen tested	Gallic acid dose (µmol)
	0	0.1	0.2
2-Nitrofluorene (1 µg)	242 ± 42	246 ± 35	245 ± 4
2-Nitro-p-phenylenediamine (30 mg)	551 ± 49	539 ± 10	470 ± 30
3-Nitro-o-phenylenediamine (300 mg)	179 ± 12	169 ± 17	160 ± 17
4-Nitro-o-phenylenediamine (10 mg)	263 ± 38	285 ± 26	300 ± 37
4,4'-Dinitro-2-biphenylamine (10 µg)	459 ± 38	549 ± 85	438 ± 3
1-Nitropyrene (0.33 µg)	465 ± 57	403 ± 18	402 ± 14
1,3-Dinitropyrene (0.0067 µg)	86 ± 11	-	82 ± 4

Values are means of revertants/plate ± SD of quadruplicate runs, each with three plates/dose.

 

 

 

Effect of tannic acid and related compounds on the mutagenicity of some aromatic amines requiring S9 in Salmonella typhimurium TA98

 

	Number of His+ revertants induced by mutagens/plate
Direct mutagen tested	Gallic acid dose (µmol)
	0	0.1	0.2
Benzidine (300 µg)	115 ± 23a	109±20a	100±8a
4-Aminobiphenyl (10 µg)	97±15c	89±21c	86±10c
3,3'-4,4'-Tetraaminobiphenyl (30 µg)	221±34e	211±12e	275±64e
N,N-N',N'-Tetramethylbenzidine (10 µg)	113±27g	114±24g	115±21g

The data are the mean ± SD from two independent runs, each with three plate/dose.

Means with different supercripts within each line group were significant different from the control.

Conclusions:

Gallic acid has been shown not to be mutagenic in the Ames Salmonella mutagenicity assay system using TA98 and TA100.
The results also indicated that gallic acid was not antimutagenic against direct mutagens such as 2-nitrofluorene, 2-nitro-p-phenylenediamine, 3-nitro-o-phenylenedia-mine, 4-nitro-o-phenylenediamine, 4,4'-dinitro-2-biphenylamine, 1-nitropyrene and 1,3-dinitropyrene using TA98.

Executive summary:

Gallic acid has been shown not to be mutagenic in the Ames Salmonella mutagenicity assay system using TA98 and TA100.

The results also indicated that gallic acid was not antimutagenic against direct mutagens such as 2-nitrofluorene, 2-nitro-p-phenylenediamine, 3-nitro-o-phenylenedia-mine, 4-nitro-o-phenylenediamine, 4,4'-dinitro-2-biphenylamine, 1-nitropyrene and 1,3-dinitropyrene using TA98.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The mutagenic activity was assayed according to Ames et al. (1975) in the modification of Yahagi et al. (1977).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Gallic acid was purchased from Koch-Light Lab. (Colnbrook, UK)

Species / strain / cell type:

S. typhimurium TA 100

Test concentrations with justification for top dose:

2.5, 5, 7.5, 10 mM

Details on test system and experimental conditions:

In the assay procedure, a mixture containing 100 µL of overnight culture (cell concentration 100--1000/nL), 25--100 µL of solutions of test chemicals and a buffer solution, in a total volume of 500 µL, was incubated for 20 min at 37 °C. For measurement of cell viability, 50 µL of each incubation mixture were taken at the end of the 20-min incubation period and diluted by a factor of 10E5 with 0.1 M Tris-HC1 buffer at pH 6.5. One hundred µL of each solution were inoculated onto maximal medium in duplicate 90-mm Petri dishes and the'number of viable cells was evaluated after 2 d at 37 °C. For mutation studies, 2.5 mL of top agar was added to 450 µL of the incubation mixture and poured onto minimal medium in 100-ram Petri dishes. Revertants were counted after 2 d of incubation at 37 °C. The decompositon rate at 37 °C of MNU and MNNG with or without the addition of gallic acid was measured at pH 5.0 and pH 6.5 using a Specord UV-VIS spectrophotometer equipped with a thermostated cuvette holder. Solutions of both components were prepared in 0.2 M citrate--phosphate buffers. The pH adjustment was done immediately before mixing the solutions and the start of measurement at zero time. At regular time intervals, minimal and maximal absorbance between 335 to 420 nm were registered. The first-rate constants and half-times were calculated from the maximum--minimum differences by the least-squares method.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not specified

Genotoxicity:

other: Inhibition of mutagenic activity of MNU and MNNG

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The dose-dependent inhibitory effects of gallic acid on the mutagenicity of 400 µM MNU and 20 µM MNNG are shown in Tables I and II, parts A. Thepresence of 10 mM gallic acid reduced the frequency of his + revertants induced by MNU to 4.5 % and by MNNG to 16.9 % of the number of MNU and MNNG-induced revertants in the absence of gallic acid. Although gallic acid was dissolved in 0.1 • Tris-HC1 buffer, its relatively high acidity and the low buffering capacity of the buffer resulted in a decrease of the pH of the
incubation mixture (containing the mutagen, gallic acid and bacteria) t(~ 4.7 (see the last column of Tables I, II, part A). The possibility that the "antimutagenic" activity of gallic acid is conditioned by its influence on the pH of the mutagenic solutions should be considered. In further experiments, we tested the antimutagenic effects of gallic acid on MNU and MNNG mutagenicity at two pH levels: pit 6.5 and pH 5.0 (Tables I, II, parts B, C). To prevent the pH decrease, we adjusted the gallic acid solution to the required pH (see Material and Methods) before adding it to the incubation mixture. The actual pH values (the last column of Tables I, II, parts B, C) did not change during 20-min incubation.

The following conclusions can be drawn (Tables I, II):
1. The mutagenic activity of MNU and MNNG is considerably higher at pH 6.5 compared to pH 5.0 (1882 versus 839 revertants after MNNG treatment). Thus, if the incubation mixture containing only MNU or MNNG has a pH of 6.5 and with l0 mM gallic acid, a pH 4.7, then the observed reduction of MNU and MNNG mutagenicity could be primarily caused by decreased pH of the incubation mixture (further referred to as nonspecific antimutagenic effect.
2. A dose-dependent inhibitory effect of gallic acid towards MNU and MNNG-induced mutagenicity appeared, however, also at controlled pH 6.5 and pH 5.0 levels of the incubation mixture, i.e. at conditions eliminating the nonspecific antimutagenic effect (Tables I, II, parts B, C). This actual antimutagenic activity of gallic acid towards MNU and MNNG mutagenicity (further referred to as specific) is less pronounced than the nonspecifie antimutagenic effect. 10 mM gallic acid reduced the number of his + revertants
induced by MNU at pH 6.5 only to 49 % and MNNG only to 47.3 % of the number of MNU and MNNG induced revertants in the absence of gallic acid. Higher doses of gallic acid than I0 mM markedly reduced cell viability and turned the agar in the plates black. 10 mM gallic acid only slightly reduced the viability in some experiments and lower doses had no influence on the viability.

The effect of timing on inhibition of MNU and MNNG mutagenicity by gallic acid is demonstrated in Table III. Preincubation of the nitrosamides with gallic acid in a bacteria-free mixture (pH 6.5) for 1 h at 37 °C led to a decrease in MNU and MNNG mutagenesis. Adding gallic acid to She top agar after 20-min MNU and MNNG treatment to the bacteria had no influence on the frequency of his + revertants (data not shown). It is, therefore, likely that the specific antimutagenic activity proceeds when phenolic acid and the mutagen are administered concurrently.

The decomposition of MNU and MNNG at pH 5.0 and 6.5 was accelerated in the presence of gallic acid (determined by the time-dependent decrease of the difference between the maximal to minimal absorbance within the measured wavelengths). The higher the concentration of gallic acid added, the shorter the half-time of the mutagen (Table IV). Whereas the maximal and minimal absorbance of MNU and MNNG solutions decreased in the absence of gallic acid, addition of gallic acid in equal concentrations or in excess of the concentration of both nitroso compounds caused an increase in absorbance especially in the.minimal region. Thus, both the polarographic and the spectrophotometric measurements revealed an interaction between the nitroso compounds and gallic acid or
their decomposition products.

Table I: Inhibition of N-methyl-N-nitrosouroa (400 µM)-induced mutagenicity ofS. typhimuriumby gallic acid

Conditions^a	Gallic acid mM	His+ revertants	% of control^b	Cell concentration l/nL	Actual pH
A	10.0	83	4.5	260	4.7
	7.5	123	6.6	320	4.9
	5.0	544	29.3	290	5.0
	2.5	1240	66.7	330	5.8
	0	1860	100	280	6.4
	0^c	113	-	310	6.4
B	10.0	929	49.0	320	6.6
	7.5	914	48.4	330	6.6
	5.0	1040	55.3	350	6.3
	2.5	1480	78.5	380	6.4
	0	1880	100	360	6.5
	0^c	142	-	430	6.5
C	10.0	308	36.7	300	5.0
	7.5	377	44.9	430	5.1
	5.0	587	67.0	330	4.9
	2.5	681	81.2	370	5.0
	0	839	100	340	5.1
	0^c	143	-	400	5.2

a A Gallic acid dissolved in Tris-HC1 buffer (pH-I 6.5) without further pH adjustment; B gallic acid dissolved in Tris-HC1 buffer and adjusted to pH 6.5; C gallic acid dissolved-in citrate--phosphate buffer and adjusted to pH 5.0.

b The number of MNU-induced revertants in the absence of gallic acid equalled 100 %

c Without MI~YU.

 

Table II: Inhibition of N-methyl-N'-nitro-N-nitrosoguanidine (20 µM)-induced mutagenicity ofS.typhimuriumby gallic acid

Conditions^a	Gallic acid mM	His+ revertants	% of control^b	Cell concentration l/nL	Actual pH
A	10.0	343	16.9	260	4.7
	7.5	779	27.9	320	4.9
	5.0	1190	38.4	270	5.2
	2.5	2170	77.9	400	5.5
	0	2710	100	330	6.3
	0^c	148	-	360	6.4
B	10.0	1060	47.3	380	6.5
	7.5	1210	54.0	450	6.6
	5.0	1410	63.0	450	6.6
	2.5	1800	80.3	400	6.5
	0	2240	100	390	6.5
	0^c	104	-	370	6.6
C	10.0	655	56.8	350	5.1
	7.5	728	63.1	430	5.0
	5.0	785	68.0	420	5.1
	2.5	894	77.5	450	5.2
	0	1150	100	420	5.0
	0^c	128	-	440	5.1

a As in Table I.

b The number of MNNG induced revertants in the absence of gallic acid equalled 100 %.

c Without MNNG.

 

Table III: Effect of preincubation of gallic acid with N-methyl-N-nitrosourea (400 µM) and N-methyl-N'-nitro-Iq-nitrosoguanidine (20 µM) without bacteria for 1 h at 37 °C on MNU and MNNG mutagenicity^a

Mutagen	Gallic acid mM	Without preincubation		With 1 h preincubation
		His+ revertants	% of control^b	His+ revertants	% of control^b
MNU	10.0	738	45.7	238^c	16.1
	7.5	874	54.1	426	28.9
	5.0	1190	73.6	824	55.9
	0	1620	100	1470	100
MNNG	10.0	1620	70.0	1090^c	45.4
	7.5	1640	71.0	1240	51.6
	5.0	1960	84.7	1490	62.0
	0	2310	100	2400	100
Control	0	165	-	134	-

a Cells added to the preincubation mixture and incubated for 20 min.

b The number of MNU and MNNG induced revertants in the absence of gallic acid equalled l00 %.

c Accompanied by a decreased cell viability.

 

Table IV: Decomposition half-times of N-methyl-N-nitrosourea (5 mM) and N-methyl-N'-nitro-N-nitrosoguanidino (5 mM) in citrate--phosphate buffers (pH 5.0 and 6.5) influenced by gallic acid^a

Nitroso compound	Gallic acid mM	Half times at 37°C, min
		pH 5.0	pH 6.5
MNU	0	602	39.7
	1	487	21.6
	5	396	13.1
	25	398	11.8
MNNG	0	919	161
	1	340	-
	5	284	57
	25	190	27

a Calculated from the differences between the maximal and minimal absorbance of the nitroso compound

Conclusions:

The frequency of his + revertants induced by N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the strain TA100 of Salmonella typhimurium was decreased by gallic and tannic acid. In weak buffer solutions, the inhibition effects of gallic acid towards MNU and MNNG mutagenicity was caused primarily by a decrease of pH in the incubation mixtures. At adjusted pH (pH 5.0 and 6.5), the antimutagenic effects are largely the result of an interaction between MNU or MNNG with phenolic acids outside the cells.

Executive summary:

The frequency of his + revertants induced by N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the strain TA100 of Salmonella typhimurium was decreased by gallic and tannic acid. In weak buffer solutions, the inhibition effects of gallic acid towards MNU and MNNG mutagenicity was caused primarily by a decrease of pH in the incubation mixtures. At adjusted pH (pH 5.0 and 6.5), the antimutagenic effects are largely the result of an interaction between MNU or MNNG with phenolic acids outside the cells.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The antimutagenic properties of various tea extracts (green tea, pauchong tea, oolong tea and black tea) and their components including (-)-epigallocatechin-3-gallate (EGCG), gallic acid and caffeine were examined by the Ames test.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Gallic acid was purchased from Sigma (St Louis, MO, USA)

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Test concentrations with justification for top dose:

10 µg/plate gallic acid

Positive controls:

yes

Positive control substance:

other: MNNG, MNU, Captan, Folpet, ICR-191, ICR-170, 9AA, 4-NQNO

Details on test system and experimental conditions:

Reverted antimutagenicity assay using the Ames test
The Ames test was carried out as described by Maron and Ames (1983), but with certain modifications. In short, we incubated the bacterial suspension, mutagen solution, tea extracts and S9 mixture at 37°C for 30 min. Then, on addition of top agar,
poured the mixture onto the surface of a minimal agar plate. After 48 hr, the number of revertant colonies were counted. Each sample was tested by using triplicate plates per test, and the data were presented as mean ± SD. The calculation of percent
inhibition as follows: Inhibition(%) = [1-(number of His+ revertants in the presence of tea extracts number of His+ revertants in the absence of tea extracts)]x100%. The number of spontaneous revertants was subtracted from the numerator and
denominator. Antimutagenic activity is defined as "stronger''-greater than 50% inhibition of mutagenic activity, ``moderate''-between 25 and 50% inhibition of mutagenic activity, and ``weaker'-less that 25% inhibition of mutagenic activity.

Preparation of S9 fraction
Liver S9 fractions were routinely prepared from Sprague-Dawley rats that were injected ip with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg. 5 days after injection, the animals were sacrificed and the livers were removed aseptically. Liver homogenates were prepared aseptically at 0±48C. Excised livers were rinsed with 0.15 M KCl, then minced and homogenized (3 ml 0.15 M KCl/g wet tissue) in a Potter-Apparatus with teflon pestle. The homogenate was centrifuged for 10 min at 9000 g at
48C. The supernatant (S9) was decanted and distributed into the freezing ampoules stored at -70°C. The microsomal enzyme reaction mix (S9 mix) was prepared immediately prior to each assay. 1 ml S9 mix has the following composition: S9, 0.1 ml; 0.04
M MgCl2, 0.02 ml; 1.65 M KCl, 0.02 ml; 0.04 M NADP, 0.1 ml; 0.05 M glucose-6-phosphate, 0.1 ml; 1.0 M NaH2PO4 (pH 7.4), 0.1 ml; distilled water, 0.56 ml.

Preparation of fresh tea extracts
Preparation of commercial tea water extracts (Wang et al., 1994). Green tea, pauchong tea, oolong tea and black tea were purchased from a local market in Taipei, Taiwan. The dry tea leaves (12.5 g) were added to 500 ml boiling water and steeped for 15 min. The infusion was cooled to room temperature and filtered. Then the aqueous tea extracts were also concentrated under vacuum and freeze-dried. The light-brown solid matter was called TWE (tea water extracts) powder.

Isolation of polyphenolic catechins from green tea leaves.
Briefly, 120 g of the fresh teas were suspended in 1 litre of distilled water (708C) and then the supernatant was collected. This step was repeated three times. The combined extracts were altered to eliminate chlorophylls and concentrated to 200 ml under reduced pressure using a rotatory vacuum evaporator. The concentrated solution was extracted with chloroform (200 ml) three times to eliminate caffeine and pigments. The remaining aqueous phase was extracted with 200 ml of ethyl acetate three times to obtain tea polyphenols. The combined ethyl acetate fractions were evaporated in a vacuum and the residue was dissolved in a small volume of water and freeze-dried. This yellow± brown solid material was called green tea polyphenols (GTP) (20% of the dry leaf weight). The compositions of GTP were determined by HPLC. GTP (20 ml, dissolved in 50% aqueous methanol) was subjected to reverse phase HPLC using a Model 600E Automated Gradient Controller (Water Associates, Milford, MA, USA) equipped with a Cosmosil 5 mm C18-MS column (4.6250 mm). Solvent systems in terms of A and B, consisted of 0.5% formic acid and methanol, respectively. The linear gradient system in terms of solvent B was: 0 min, 15%; 0±15 min, a linear increase to 20%;
15±40 min, a linear increase to 40%; 40±70 min, a linear increase to 50%. The GTP constituents were eluted at a ¯ow rate of 1 ml/min and monitored at 280 nm using a Waters Module 484 absorbance detector (Waters Associates, Milford, MA, USA).

Determination of the mechanism of antimutagenic action of EGCG and gallic acid
(I) Blank: 0.1 ml MNNG (1 mg/plate) or 9AA (10 mg/plate) were incubated with 0.8 ml 0.1 M phosphate buffer, and 0.1 ml cell suspension at 37°C for 30 min, without antimutagen (blank).
(II) Mutagen then antimutagen: 0.7 ml 0.1 M phosphate buffer (pH 7.4) and 0.1 ml bacterial cell suspension were added with 0.1 ml MNNG (1 mg/plate) or 9AA (10 mg/plate) at 37°C for 15 min, then washed once with phosphate buffer and incubated with 0.1 ml EGCG (1 mg/plate) or gallic acid (10 mg/plate) at 37°C for 15 min.
(III) Antimutagen then mutagen: 0.7 ml 0.1 M phosphate buffer (pH 7.4) and 0.1 ml bacterial cell suspension were preincubated with 0.1 ml EGCG (1 mg/plate) or gallic acid (10 mg/plate) at 37°C for 15 min, then washed once with phosphate buffer and incubated with 0.1 ml MNNG (1 mg/plate) or 9AA (10 mg/plate) at 37°C for 15 min.
(IV) All components then bacteria: 0.1 ml MNNG (1 mg/plate) or 9AA (10 mg/plate) were preincubated with 0.1 ml EGCG (1 mg/plate) or gallic acid (10 mg/plate) in 0.7 ml phosphate buffer at 37°C for 15 min, then 0.1 ml of the cell suspension was added and the mixture was incubated at 37°C for 15 min.
(V) All components simultaneously: 0.1 ml MNNG (1 mg/plate) or 9AA (10 mg/plate), 0.1 ml EGCG (1 mg/plate) or gallic acid (10 mg/plate) and 0.1 ml of the cell suspension were simultaneously incubated in 0.7 ml of phosphate buffer at 37°C for 30 min.

Evaluation criteria:

Antimutagenic activity is defined as "stronger''-greater than 50% inhibition of mutagenic activity, "moderate''-between 25 and 50% inhibition of mutagenic activity, and ``weaker'-less that 25% inhibition of mutagenic activity.

Species / strain:

S. typhimurium, other: TA 97, 98 & 100

Metabolic activation:

not specified

Genotoxicity:

other: Antimutagenic action

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The antimutagenic activity of the components of tea in pure form are shown in Tables 1 and 2. The three major components of tea extracts, EGCG, gallic acid and caffeine, were examined for antimutagenic activity. the antimutagenic activity of these
components against the mutagenicity of MNNG, MNU captain and folpet in strain TA100 showed a decreasing pattern as follows: EGCG>gallic acid>caffeine. At the highest dose of EGCG (100 mg/plate), the mutagenicities of MNNG, MNU, captan and folpet were inhibited by up to 85%, whereas the mutagenicities of MNNG, captan and folpet were inhibited by gallic acid up to 60%. However, caffeine showed a weaker but significantly inhibitory effect.
When the antimutagenic potential of the three components of tea extracts, EGCG, gallic acid and caffeine, were evaluated against the direct-acting carcinogen 9-aminoacridine (9AA) in strain TA98, a decrease in mutagenicity was observed. But caffeine was the least effective. Nevertheless, gallic acid showed a greater inhibitory effect against 9AA, and EGCG was slightly less effective than gallic acid in strain TA98.
In strain TA97, the inhibitory effect of three major components of tea extract against 4-NQNO, ICR-191 and ICR-170 decreased as follows: EGCG>gallic acid>caffeine. The EGCG appeared to inhibit the mutagenicity of the indirect-acting mutagens of BP more significantly than gallic acid, but EGCG was less effective than gallic acid when tested with other indirect-acting mutagens, AFB1 and AAF in strain TA98.

In order to determine the mechanism of the antimutagenic action of EGCG and gallic acid, the cells were subjected to five different treatments with MNNG or 9AA. EGCG and gallic acid showed antimutagenic effect in various treatments. Among these various
treatments the greatest inhibition of MNNG-induced mutagenesis was found when the MNNG and EGCG were pre-incubated together before exposing to bacteria TA100. The significant difference between the action of EGCG and gallic acid action was observed. When the bacteria and gallic acid were pre-incubated together before exposure to 9AA in strain TA98, gallic acid displayed a pronounced antimutagenic activity, and the strongest inhibitory effect of 9AA-induced mutagenic activity was observed.

Table 1: Antimutagenic effects of gallic acid to direct-acting mutagens in Salmonella strains TA100, TA97 and TA98

		His^+revertant/plate^a
Mutagens	Dose (µg/plate)	Gallic acid	Strains
MNNG (1 µg/plate)	Control^b	1109±218(-)^c,d	TA100
	0.1	810±75(26%)
	1.0	765±91(31%)
	10	710±50(36%)
	100	410±37(63%)
MNU (10 mg/plate)	Control	2174±192(-)	TA100
	0.1	2154±149(1%)
	1.0	2046±108(6%)
	10	1903±267(13%)
	100	1785±95(18%)
Captan (100 mg/plate)	Control	813±34(-)	TA100
	0.1	582±35(28%)
	1.0	520±74(36%)
	10	422±48(48%)
	100	398±47(51%)
Folpet (100 mg/plate)	Control	245±9(-)	TA97
	0.1	196±42(20%)
	1.0	169±29(31%)
	10	167±16(32%)
	100	89±23(64%)
ICR-191 (10 mg/plate)	Control	3036±192(-)	TA97
	0.1	2944±178(3%)
	1.0	2914±252(4%)
	10	2762±310(9%)
	100	2064±148(32%)
ICR-170 (10 mg/plate)	Control	2979±281(-)	TA97
	0.1	2919±277(2%)
	1.0	2859±350(4%)
	10	2710±392(9%)
	100	2651±127(11%)
9AA(10 mg/plate)	Control	658±39(-)	TA98
	0.1	210±17(68%)
	1.0	165±22(75%)
	10	125±9(81%)
	100	112±19(83%)
4-NQNO(10 mg/plate)	Control	563±66(-)	TA97
	0.1	534±59(5%)
	1.0	428±39(24%)
	10	377±67(33%)
	100	320±74(43%)

a Values are the mean±SD number of histidine revertants from three plates from two independant experiments. The number of induced revertants have been corrected for the spontaneous reversion rate of TA100 (107±26), TA98 (26±6) and TA97 (102±4), respectively.

b The number of controls was determined without tea extract.

c Inhibition (%) =[1-(number of His+ revertants in the presence of tea extracts / number of His+ revertants in the absence of tea extracts)]x100%.

d Data presented are the mean of three experiments.

 

Table 2: Antimutagenic effects of gallic acid to indirect-acting mutagens in Salmonella strain TA98

		His^+revertant/plate^a
Mutagens	Dose (µg/plate)	Gallic acid	Strains
AAF (10 mg/plate) (+S9)	Control^b	634±60(-)^c,d	TA98
	0.1	608±59(4%)
	1.0	590±39(7%)
	10	553±67(13%)
	100	507±74(20%)
BP (10 mg/plate) (+S9)	Control	521±66(-)	TA98
	0.1	471±59(10%)
	1.0	437±39(16%)
	10	430±67(17%)
	100	415±74(20%)
AFB1 (1 mg/plate) (+S9)	Control	617±66(-)	TA98
	0.1	614±59(1%)
	1.0	600±39(3%)
	10	505±67(18%)
	100	431±74(20%)

a Values are the mean±SD number of histidine revertants from three plates from two independent experiments. The number of induced revertants have been corrected for the spontaneous reversion rate of TA98 (26±6).

b The number of controls was determined without tea extract.

c Inhibition (%) = [1-(number of His+ revertants in the presence of tea extracts/number of His+ revertants in the absence of tea extracts)]x100%.

d Data presented are the mean of three experiments.

Conclusions:

The antimutagenic properties of various tea extracts (green tea, pauchong tea, oolong tea and black tea) and their components including (-)-epigallocatechin-3-gallate (EGCG), gallic acid and caffeine were examined by the Ames test (TA100, TA98 and TA97). Gallic acid, the major component of black tea strongly inhibited the mutagenicity of 9AA, and moderately inhibited the mutagenicity of MNNG and folpet. Gallic acid perhaps could act as nucleophiles to scavenge the electrophilic mutagens.

Executive summary:

The three major components of tea extracts, EGCG, gallic acid and caffeine, were examined for antimutagenic activity. The antimutagenic activity of these components against the mutagenicity of MNNG, MNU captain and folpet in strain TA100 showed a decreasing pattern as follows: EGCG>gallic acid>caffeine. The mutagenicities of MNNG, captan and folpet were inhibited by gallic acid up to 60%.

When the antimutagenic potential of the three components of tea extracts, EGCG, gallic acid and caffeine, were evaluated against the direct-acting carcinogen 9-aminoacridine (9AA) in strain TA98, a decrease in mutagenicity was observed. Gallic acid showed a greater inhibitory effect against 9AA.

In strain TA97, the inhibitory effect of three major components of tea extract against 4-NQNO, ICR-191 and ICR-170 decreased as follows: EGCG>gallic acid>caffeine.

In order to determine the mechanism of the antimutagenic action of EGCG and gallic acid, the cells were subjected to five different treatments with MNNG or 9AA. EGCG and gallic acid showed antimutagenic effect in various treatments. Among these various treatments the greatest inhibition of MNNG-induced mutagenesis was found when the MNNG and EGCG were pre-incubated together before exposing to bacteria TA100. The significant difference between the action of EGCG and gallic acid action was observed. When the bacteria and gallic acid were pre-incubated together before exposure to 9AA in strain TA98, gallic acid displayed a pronounced antimutagenic activity, and the strongest inhibitory effect of 9AA-induced mutagenic activity was observed.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Bacterial reversion assay called the WP2 Mutoxitest (based on strain IC203, deficient in OxyR, and its oxyRq parent WP2 uvrA/pKM101 (denoted IC188)) has been used in the evaluation of gallic acid for oxidative mutagenicity.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Gallic acid (3,4,5-trihydroxybenzoic acid)
Code number (37)
CAS 149-91-7
Source: Sigma

Target gene:

WP2 mutagenicity test

Species / strain / cell type:

bacteria, other: IC203

Additional strain / cell type characteristics:

other: a derivative of WP2 uvrA/pKM101 deficient in the OxyR function

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

E. coli, other: IC204

Additional strain / cell type characteristics:

other: a derivative of WP2 uvrA carrying a DumuDC::cat mutation

Species / strain / cell type:

E. coli, other: IC206

Additional strain / cell type characteristics:

other: a mutY::kan derivative of IC204

Species / strain / cell type:

E. coli, other: IC208

Additional strain / cell type characteristics:

other: DoxyR30 derivative of IC206

Test concentrations with justification for top dose:

500, 750, 1000 µg/plate

Vehicle / solvent:

DMSO: 200 mg/ml., then water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

Media
Nutrient broth was Oxoid Nutrient Broth No. 2. LA medium contained 5 g NaCl, 10 g Difco Bactotryptone, 5 g Difco yeast extract and 20 g Difco agarper litre of distilled water. Solid minimal E4 medium contained 15 g Difco agar and 4 g glucose per litre
of Vogel-Bonner E medium. ET4 medium was solid minimal E4 medium supplemented with 0.5 mg tryptophan per litre. Top agar contained either 6 g Difco agar and 5 g NaCl per litre of distilled water for mutagenicity assays or 7.5 g Difco agar per litre
of distilled water for cytotoxicity tests. S9 liver homogenate was prepared from uninduced rats as described.

Plate reverse mutation assays
Plate incorporation assays were performed by mixing 100 ml of broth cultures 100 ml from frozen permanents inoculated into 10 ml of nutrient broth and incubated overnight at 378C. of each tester strain, 100 ml of a suitable dilution of the test compound, 50 ml of S9 (when indicated). and 2.5 ml of molten top agar. The mixture was poured on minimal ET4 plates. These were incubated 2 days at 378C. In assays with hypoxanthine and xanthine, 0.1 U per plate of xanthine oxidase Sigma. were added. In assays with ascorbic acid, an ascorbic acid cupric sulfate solution molar ratio 100:1. was used. To evaluate the number of preplating mutants originated during the overnight growth, the zero dose in the mutagenesis assay was screened on unsupplemented E4 plates. Nutrient broth was supplemented with 20 mgrml ampicillin for the overnight cultures of strains containing pKM101. Each compound was tested at least twice with five to six dose levels, including a toxic dose, and the means of Trpq revertants per plate are presented. A positive result is defined as a reproducible, dose-related increase in the number of revertants. The increase should reach at least a doubling of the number of spontaneous revertants.

Cytotoxicity tests
For growth inhibition experiments, 100 ml of
overnight broth cultures and 50 ml of S9 (when indicated) were added to 3 ml of molten top agar and poured on LA plates. Paper discs (6 mm in diameter) were impregnated with 10 ml of solutions containing test compounds, placed on the solidified top agar plates and allowed to incubate overnight. The diameter of the zone of inhibition (in mm) was obtained by measuring the diameter (including the disc) of the zone and subtracting the diameter of the disc.

Evaluation criteria:

Plate reverse mutation assays
A positive result is defined as a reproducible, dose-related increase in the number of revertants. The increase should reach at least a doubling of the number of spontaneous revertants. In the WP2 Mutoxitest, a mutagenic response in IC203 greater than in WP2 uvrA/pKM101 (here denoted IC188). is interpreted as an indicator of oxidative mutagenesis.

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

E. coli, other: IC203

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

without S9

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The presence of S9 in assays with IC203 had a protective effect

Mutagenicity results of gallic acid in the WP2 Mutoxitest

			Number of revertants/plate
Compound	Dose (µg/plate)	Test result	IC188	IC203	IC203+S9
-	-		145	144	151
Gallic acid (mg)	500	oxidative mutagen	175	200	175
	750		160	302	192
	1000		165	616	160

 

Cytotoxic effect of oxidants: influence of rat liver S9

Compound	Dose (µg/disc)	Inhibition (mm)
		IC188	IC203	IC203 + S9
Gallic acid	1000	3	18	3

 

Induction of 8-oxoguanine-promoted mutations by the 31 oxidative mutagens in MutY-deficient strains

Compound	Dose (µg/plate)	Number of revertants / plate
		IC204 (mut+)	IC206 (mutY)	IC208 (mutY oxyR)
-	-	10	28	32
Gallic acid	1000	8	21	34

Conclusions:

Bacterial reversion assay called the WP2 Mutoxitest (based on strain IC203, deficient in OxyR, and its oxyRq parent WP2 uvrA/pKM101 (denoted IC188)) has been used in the evaluation of gallic acid (among 80 chemicals) for oxidative mutagenicity. Gallic acid caused oxidative mutagenesis, being positive only in IC203 and sensitive to inhibition by S9.

Executive summary:

Bacterial reversion assay called the WP2 Mutoxitest (based on strain IC203, deficient in OxyR, and its oxyRq parent WP2 uvrA/pKM101 (denoted IC188)) has been used in the evaluation of gallic acid (among 80 chemicals) for oxidative mutagenicity. Gallic acid caused oxidative mutagenesis, being positive only in IC203 and sensitive to inhibition by S9.

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Antioxidant activity was evaluated by using four different test systems named as β-carotene bleaching, DPPH free radical scavenging, reducing power and chelating effect.

GLP compliance:

not specified

Remarks:

published data without specification of GLP compliance

Type of assay:

other: antioxidant and DNA damage protection potential

Test concentrations with justification for top dose:

0.1, 0.2, 0.3, 0.5 mg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Butylated hydroxytoluene (BHT), Butylated hydroxyanisole (BHA), EDTA

Details on test system and experimental conditions:

Total antioxidant activity by β-Carotene–linoleic acid method
In this assay antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation (Aktumsek et al., 2013). A stock solution of β-carotene–
linoleic acid mixture was prepared as following: 0.5mg β-carotene was dissolved in 1ml of chloroform (HPLC grade). Twenty-five microliters of linoleic acid and 200 mg
Tween 40 were added. Chloroform was completely evaporated using a vacuum evaporator. Then 100 ml of oxygenated distilled water was added with vigorous shaking;
2.5ml of this reaction mixture was dispersed to test tubes and 0.5ml of the extracts (2.0 mg/ml) in water were added and the emulsion system was incubated for up to
2 h at 50 °C. The same procedure was repeated with the positive control BHT, BHA and a blank. After this incubation period, absorbance of the mixtures was measured
at 490 nm. Measurement of absorbance was continued until the color of β-carotene disappeared. The bleaching rate (R) of β-carotene was calculated according to Eq. (1).
R = ln(a b) t (1)
where, ln = natural log, a = absorbance at time 0, b = absorbance at time t (120 min) (Aktumsek et al., 2013). The antioxidant activity (AA, %) was calculated in terms of percent inhibition relative to the control using Eq. (2).
AA = [(Rcontrol −Rsample) Rcontrol]×100 (2)
Antioxidative activities of the extracts were compared with those of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) at 2.0 mg/ml and blank consisting of only 0.5 ml water.

Scavenging effect on 1,1-Diphenyl-2-picrylhydrazyl (DPPH)
The hydrogen atoms or electrons donation abilities of the corresponding compounds were measured from the bleaching of purple colored methanol solution of DPPH (Aktumsek et al., 2013). One milliliter of various concentrations (0.2–1.0 mg/
ml) of the extracts in water was added to a 1 ml of DPPH radical solution in methanol (final concentration of DPPH was 0.2 mM). The mixture was shaken vigorously and
allowed to stand for 30 min; the absorbance of the resulting solution was measured at 517 nm with a spectrophotometer (Shimadzu UV-1601, Kyoto, Japan).
Inhibition of free radical DPPH in percent (I %) was calculated in the following way:
I% = 100 × (AControl − ASample ) AControl
where, AControl is the absorbance of the control reaction (containing all reagents except the test compound), and ASample is the absorbance of the test compound. BHT and BHA were used as a control.

Reducing power
The reducing power was determined according to the method of Aktumsek et al. (2013). Each extract (0.2–1.0 mg/ml) in water (2.5 ml) was mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide
and the mixture was incubated at 50 °C for 20 min. Then, 2.5 ml of 10% trichloroacetic acidwere added, and the mixturewas centrifuged at 200 g (MSE Mistral 2000, London, UK) for 10 min. The upper layer (2.5 ml) was mixed with 2.5 ml of
deionized water and 0.5 ml of 0.1% ferric chloride. Finally the absorbance was measured at 700 nm against a blank. BHT and BHA were used as a control.

Chelating effects on ferrous ions
The chelating effect was determined according to the method of Aktumsek et al. (2013). Briefly, 1 ml (2 mg/ml) of the extracts in water was added to 1 ml of water and a solution of 2 mM FeCl2 (0.05 ml). The reaction was initiated by the addition
of 5mMferrozine (0.2 ml). Then, the mixture was shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was measured spectrophotometrically at 562 nm. The inhibition percentage of ferrozine–Fe2+ complex formation
was calculated by using the formula given below:
Metal chelating effect (%) = [(AControl − ASample ) AControl ]×100
where AControl is the absorbance of control (the control contains FeCl2 and ferrozine, complex formation molecules) and ASample is the absorbance of the test compound. EDTA was used as a control.

DNA damage protection potential
DNA damage protection activities of the extracts were evaluated on pBR322 plasmid DNA (Vivantis). Plasmid DNA was oxidized with H2O2 + UV treatment in the presence of extracts and checked on 1% agarose gels according to Tepe et al. (2011).
In brief, the experiments were performed in a volume of 10 μl in a microfuge tube containing 3 μl pBR322 plasmid DNA (172 ng/μl), 1 μl of 30% H2O2, and 5 μl of extract in the concentrations of 5, 10, 15 and 20 mg/ml, respectively. The reactions were
initiated by UV irradiation and continued for 5 min on the surface of a UV transilluminator (DNR-IS) with an intensity of 8000 μW/cm2 at 302 nm at room temperature. After irradiation, the reaction mixture (10 μl) along with gel loading dye (6×) was
loaded on a 1% agarose gel for electrophoresis. Untreated pBR322 plasmid DNA was used as a control in each run of gel electrophoresis along with partially treated plasmid, i.e. only UV or only H2O2 treatment. Gels were stained with EtBr and photographed with the Gel documentation system (DNR-IS, MiniBIS Pro).

Statistics:

All assays were carried out in triplicate for all the experiments. The results are expressed as mean and standard deviation values (mean ± SD). Differences between means were determined by the analysis of variance (ANOVA) with Tukey’s honestly
significant difference post hoc test with α = 0.05, which were analyzed with SPSS v. 14.0.

Species / strain:

other: not relevant

Additional information on results:

Total antioxidant activities of the phenolic acids measured by β-carotene bleaching method are presented in Table 1. As can be seen from the table, gallic acid showed lower activity than those of positive controls, BHT and BHA.

Scavenging abilities of the phenolic acids on DPPH free radical were also tested (Table 2). None of the phenolic acids tested at 0.1 mg/ml concentration showed activity as high as positive controls, BHT and BHA. Additionally, at 0.1 mg/ml concentration, free
radical scavenging ability of BHT was still higher than those of phenolic acids. At 0.5 mg/ml concentration, majority of phenolic acids were found active on DPPH free radical except chlorogenic, protocatechuic, and gallic acids. Gallic acid at 0.5 mg/ml exhibited a scavenging activity potential in this test system of 73.62%.

Phenolic acids were also tested for their reducing power potentials by the method of Aktumsek et al. (2013) at three different concentration levels (Table 3). At all concentration values, none of phenolic acids showed activity as high as BHT. At 0.1 and 0.3 mg/ml, gallic acid showed lower activity than that of BHA. At 0.5 mg/ml, gallic acid showed higher activity than that of BHA. In this test system, gallic acid at 0.5 mg/ml showed the weakest activity potential (1.407). Results obtained from this system were found to be statistically different except ferulic, syringic, and vanillic acids.

EDTA was used as positive control agent to determine the chelating effects of phenolic acids on ferrous ions. According to the results presented in Table 4, none of phytochemicals were active as EDTA. Chelating effect of gallic acid was determined as 38.7%, lower than that of EDTA (98.4%). Results obtained from gallic, and p-hydroxybenzoic acids are found similar from the statistical point of view.

Phenolic acids listed above were subjected to a test to determine their protective effect on pBR322 plasmid DNA against the toxic effects of UV and H2O2, which are highly mutagenic on DNA. Results obtained from this experiment are given in Table 5.
DNA derived from pBR322 plasmid showed three bands on agarose gel electrophoresis; the faster moving band corresponded to the native form of supercoiled circular DNA (scDNA), the slower moving band was the open circular form (ocDNA), and linear DNA (lnDNA), which is the result of the cleavage of supercoiled circular DNA arisen from the UV photolysis of H2O2. OH produced from the UV photolysis of H2O2 forms DNA strand breakage and smears are seen on the gel.

As can be seen from Table 5, all of the samples showing activity have protected the slowermoving band (ocDNA) of plasmid DNA. Phenolic acids showed lower protective effect on scDNA when compared to the other bands. Gallic acid (at 0.002 mg/ml),
could not protect pBR322 plasmid DNA.

According to literature data, gallic acid has also DNA damage protection potential. Nair and Nair (2013) have studied the radioprotective effect of gallic acid in mice. According to this report, one hour prior to whole body gamma radiation exposure, the compound
reduced the radiation-induced cellular damage in peripheral blood leukocytes, bone marrow cells, and splenocytes. According to another report, gallic acid consumption reduced the DNA damage caused by treatment of the cells with reactive oxygen species at a
rate of 41% (Ferk et al., 2011). Although gallic acid has protective effect on DNA, some researchers showed the damage provoking effect of this compound on genetic material in several cancer cell lines such as human prostate cancer PC-3 and HeLa cells (Erol-Dayi et al., 2012; Liu et al., 2013). On the other hand, according to some reports, presence of gallic acid induces single and double strand breaks in plasmid DNA in free cell systems (Biso et al., 2010; Matsuda and Nakajima, 2012).

Remarks on result:

other: Antioxidant activity was evaluated

Table 1 : Total antioxidant activities of gallic acid byβ-carotene bleaching method^a.

Phenolic acids	Total antioxidant activity (%)^b
Gallic acid	82.30 ± 0.54****
Positive control: BHT	86.48 ± 1.93**
Positive control: BHA	92.14 ± 0.15***

aValues expressed are means±S.D. of three parallel measurements; in the same column, data marked with different numbers of superscript symbols indicate significant difference (p<0.05).

bAt 2.0 mg/ml concentration.

 

Table 2 : Radical scavenging activities of phenolic acid.^a.

Phenolic acids	DPPH free radical scavenging activity (%)
	0.1 mg/ml	0.3 mg/ml	0.5 mg/ml
Gallic acid	47.19 ± 1.17****	68.24 ± 1.79****	73.62 ± 0.39***
Positive control: BHT	87.14 ± 0.62*******	-	-
Positive control: BHA	79.40 ± 0.13********	-	-

aValues expressed are means±S.D. of three parallel measurements; in the same column, data marked with different

numbers of superscript symbols indicate significant difference (p<0.05).

 

 

Table 3 : Reducing power potentials of phenolic acids.^a.

Phenolic acids	Reducing power potential (absorbance at 700 nm)
	0.1 mg/ml	0.3 mg/ml	0.5 mg/ml
Gallic acid	0.547 ± 0.022****	0.902 ± 0.011****	1.407 ± 0.056*****
Positive control: BHT	2.012 ± 0.029****	-	-
Positive control: BHA	1.114 ± 0.098*****	-	-

aValues expressed are means±S.D. of three parallel measurements; in the same column, data marked with different

numbers of superscript symbols indicate significant difference (p<0.05).

 

Table 4 : Chelating effects of phenolic acids.^a.

Phenolic acids	Chelating effect (%)
Gallic acid	38.70 ± 0.72***
Positive control: EDTA	98.40 ± 0.35*******

aValues expressed are means±S.D. of three parallel measurements; in the same column, data marked with different numbers of superscript symbols indicate significant difference (p<0.05).

 

Table 5 : A clear overview to the protected and non-protected bands of pBR322 plasmid DNA.

Phenolic acids	Concentration (mg/ml)	ocDNA^a	lnDNA^b	scDNA^c
Gallic acid	0.002	-	-	-
	0.004	+	-	-
	0.008	+	-	-
	0.016	+	+	-
	0.032	+	+	-

aocDNA: Open circular DNA band.

blnDNA: Linear DNA band.

cscDNA: Super coiled DNA band.

Conclusions:

In this study, gallic acids was evaluated among other phenolic acids for its antioxidant and DNA damage protection potentials. Antioxidant activity was evaluated by using four different test systems named as β-carotene bleaching, DPPH free radical scavenging, reducing power and chelating effect. Antioxidative activities of the extracts were compared with those of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) used as positive controls.

In bleaching method, total antioxydant activity of gallic acid is lower than activity of positive controls (with significant difference).
DPPH free radical scavenging activity of gallic acid is lower than activity of positive controls (with significant difference).
Gallic acid was also tested for its reducing power potentials at three different concentration levels. At all concentration values, gallic acid did not show activity as high as BHT but exhibited higher activity than BHA.
EDTA was used as positive control agent to determine the chelating effects of gallic acid on ferrous ions. Gallic acid was not active as EDTA.
Gallic acid was subjected to a test to determine its protective effect on pBR322 plasmid DNA against the toxic effects of UV and H2O2, which are highly mutagenic on DNA. Gallic acid could not protect pBR322 plasmid DNA.

Executive summary:

In this study, gallic acids was evaluated among other phenolic acids for its antioxidant and DNA damage protection potentials. Antioxidant activity was evaluated by using four different test systems named as ?-carotene bleaching, DPPH free radical scavenging, reducing power and chelating effect. Antioxidative activities of the extracts were compared with those of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) used as positive controls.

In bleaching method, total antioxydant activity of gallic acid is lower than activity of positive controls (with significant difference).

DPPH free radical scavenging activity of gallic acid is lower than activity of positive controls (with significant difference).

Gallic acid was also tested for its reducing power potentials at three different concentration levels. At all concentration values, gallic acid did not show activity as high as BHT but exhibited higher activity than BHA.

EDTA was used as positive control agent to determine the chelating effects of gallic acid on ferrous ions. Gallic acid was not active as EDTA.

Gallic acid was subjected to a test to determine its protective effect on pBR322 plasmid DNA against the toxic effects of UV and H2O2, which are highly mutagenic on DNA. Gallic acid could not protect pBR322 plasmid DNA.

Reference 7

Endpoint:

genetic toxicity in vitro, other

Remarks:

Antimutagenic mechanism of action

Type of information:

other: Review

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Explanation of mode of action of gallic acid as mutagen

GLP compliance:

no

Type of assay:

bacterial forward mutation assay

Species / strain / cell type:

not specified

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Remarks on result:

other: not specified

Conclusions:

Antimutagenic effect of gallic acid has been tested by the Ames test. This compound could perhaps act as a nucleophile to scavenge the electrophilic mutagens. Moreover, it was implied that gallic acid can bind or insert into the outer membrane transporters and lead to the blockage of a mutagen that was transferred into the cytosol.

Executive summary:

Antimutagenic effect of gallic acid  has been tested by the Ames test. This compound could perhaps act as a nucleophile to scavenge the electrophilic mutagens. Moreover, it was implied that gallic acid can bind or insert into the outer membrane transporters and lead to the blockage of a mutagen that was transferred into the cytosol.

Reference 8

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study, we examined whether the six tea constituents, namely (−)-epigallocatechin-3-O-gallate (EGCg), (−)-epicatechin-3-O-gallate (ECg), (−)-epigallocatechin (EGC), (−)-epicatechin (EC), (+)-catechin (+C) and gallic acid (GA), can induce chromosomal damage by themselves, and/or whether they can protect against ROS-induced chromosomal damage at their physiological concentrations after drinking normal amounts of tea. Chromosomal damage was evaluated by cytokinesis-block micronucleus assay using WIL2-NS cells, a sensitive detection system for ROS-induced chromosomal damage.

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Gallic acid obtained from Funakoshi Co., Ltd. (Tokyo, Japan).

Target gene:

WIL2-NS cells

Species / strain / cell type:

mammalian cell line, other:

Details on mammalian cell type (if applicable):

WIL2-NS cells (ATCC No. CRL-8155), a non-immunoglobulin secreting human B lymphocyte line established from the spleen of a Caucasian male with hereditary spherocytic anemia, were obtained from the American Type Culture Collection (Manassas, VA, USA).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive control substance:

other: H2O2, tert-butyl hydroperoxide(tert-BuOOH)

Details on test system and experimental conditions:

Cell culture and treatment with catechins and oxidants
WIL2-NS cells (ATCC No. CRL-8155), a non-immunoglobulin secreting human B lymphocyte line established from the spleen of a Caucasian male with hereditary spherocytic anemia, were obtained from the American Type Culture Collection (Manassas, VA,
USA). The cells were reported to be highly sensitive to chromosomal damage detection induced by ROS, particularly H2O2. Cell culture and treatment with catechins and oxidants were performed as described elsewhere. Briefly, WIL2-NS cells were
cultured and maintained. The WIL2-NS cells were washed once with HBSS, and resuspended in HBSS at a density of 0.5 × 106 cells per ml. Cell suspensions (950 µl) were incubated for 60 min with various concentrations of tea catechins (in 50µl) dissolved in HBSS. To determine whether tea catechins induced chromosomal damage, the cells were washed with HBSS after treatment with or without tea catechins, and then subjected to the CBMN assay as described below. To evaluate the protective effects by tea catechins on ROS-induced chromosomal damage, the cell suspensions with and without the tea catechins treatment were exposed to either H2O2 or tert-BuOOH at 37 ◦C for 30 min, washed with HBSS, and then subjected to the CBMN assay.

Analytical methods
Chromosomal damage in WIL2-NS cells was assessed by the CBMN-assay. Briefly, WIL2-NS cells were washed with HBSS and with RPMI 1640 to remove catechins and/or oxidants, then resuspended in RPMI 1640 medium containing 10% (v/v) fetal bovine serum, 1% (v/v) antibiotic solution, 2mM glutamine, 4.44 µg/ml cytochalasin B at a cell density of 0.5 × 106 cells per ml. After 42 h of culture, the cells were harvested. Slides were prepared using a cytocentrifuge (Shandon Southern Products, Cheshire, UK), air dried, and fixed with absolute methanol, and then stained with 4% (v/v) Giemsa’s solution in water for 30 min. Chromosomal damage rates were expressed as the number of micronucleated binucleate cells (MNed BN cells) per 1000 binucleated cells (BN cells), and the nuclear division index (NDI) was calculated as reported previously. H2O2 generation by tea catechins was measured by the phenol red method with H2O2 as a standard. Each tea catechin was dissolved in HBSS immediately before use.

Statistics:

The data are presented as mean ± S.E. for triplicate experiments. Statistical analyses of the data were carried out using ANOVA followed by a post hoc test of Fisher’s protected least significant difference. A P-value <0.05 was considered to be significant. The statistical analyses were performed using a computer program (StatView ver. 5.0, Abacus Concepts, CA, USA).

Species / strain:

mammalian cell line, other: WIL2-NS cells

Metabolic activation:

not applicable

Genotoxicity:

negative

Remarks:

anti-genotoxic

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

An induction of chromosomal damage by the catechins themselves was evaluated in WIL2-NS. In this experiment, the effect of gallic acid, another component of tea, was tested. Concentrations of each tea catechin in blood after taking excess
tea catechins was reported to be less than 10 µM, and in the present experiment production of H2O2 by each tea catechin in vitro was almost undetectable at <10µM.

Based on these findings, WIL2-NS cells were treated with 1 µM or 10 µM of tea constituent, and cytostatic and chromosomal damage by the tea constituent was examined by CBMN assay. Gallic acid did not cause a delay in cell division (Table 1).

Although tert-BuOOH (500 µM) and H2O2 (30µM), two positive controls, induced significant chromosomal damage, gallic acid at <10µM did not induce chromosomal damage.

To examine the preventive effect of tea constituents against reactive oxygen species (ROS)-induced cytostatic effect and chromosomal damage, WIL2-NS cells were pretreated with various concentrations of tea catechins and GA for 60 min, and then exposed to H2O2 or t-BuOOH for 30 min. Nuclear division index of either 30 µM H2O2 or 500µM t-BuOOH treatment was not significantly influenced by the treatment of 0.3 to 10 µM of tea constituents (including GA) (Table 2). In contrast, each tea constituent (including GA), except EC, prevented the H2O2-induced chromosomal damage even at the low concentration of 1µM. EGC and GA did not prevent this damage even at a concentration of 10µM.

Table 1 : Cytostatic and genotoxic effect of various tea constituents in WIL2-NS cells

Cells	Concentration (µM)	Nuclear division index	MNed BN per 1000 BN cells
Untreated control		2.4 ± 0.01	8.6 ± 2.1
Positive control:
H2O2	30	2.1 ± 0.07^a	81.1 ± 15.4^a
t-BuOOH	500	2.2 ± 0.01^a	81.4 ± 5.9^a
Gallic acid	1	2.4 ± 0.10	8.0 ± 3.7
	10	2.4 ± 0.05	8.4 ± 2.5

aSignificance for comparison to untreated control groups,P <0.05.

 

WIL2-NS cells were treated with various concentrations of tea constituents (1, 10µM) for 60 min. The cells were subjected to CBMN assay to determine cytostatic (nuclear division index) and genotoxic effects (MNed BN cells). Values are mean and S.E.M. for triplicate determination from separate cultures.

 

Table 2 : Effects of catechins on nuclear division index in ROS-exposed WIL2-NS cells

	Catechin concentration (µM)	Nuclear division index
		H2O2(30 µM)	t-BuOOH (500 µM)
Control	0	2.1 ± 0.07	2.2 ± 0.01
Gallic acid	0.3	2.4 ± 0.09	2.4 ± 0.10
	1	2.2 ± 0.05	2.2 ± 0.06
	10	2.2 ± 0.06	2.3 ± 0.10

 

WIL2-NS cells were incubated with tea catechins (0, 0.3, 1, 10 µM) at 37 ◦C, for 60 min and then were exposed to reactive oxygen species (30 µM of H2O2, 500µM of tert-BuOOH) for 30 min. The cells were subjected to CBMN assay to evaluate cytostatic effects (nuclear division index). Values are mean and S.E.M. for triplicate determination from separate cultures.

Conclusions:

Using the cytokinesis-block micronucleus assay in WIL2-NS cells, the authors investigated the effects of six tea constituents, including gallic acid (GA), on chromosomal damage in two ways; induction by each component on its own and prevention against treatment of reactive oxygen species (ROS). None of the tea constituents induced chromosomal damage at <10µM. On the other hand, GA prevented H2O2-induced chromosomal damage in a dose-dependent manner with a significant effect detected at 1µM. Chromosomal damage induced by tert-butylhydroperoxide was not prevented by GA even at 10µM. These results suggest that physiological concentration of tea constituents are not genotoxic but rather anti-genotoxic against ROS, although their preventive effects are slightly different depending on their chemical structure.

Executive summary:

Using the cytokinesis-block micronucleus assay in WIL2-NS cells, the authors investigated the effects of six tea constituents, including gallic acid (GA), on chromosomal damage in two ways; induction by each component on its own and prevention against treatment of reactive oxygen species (ROS). None of the tea constituents induced chromosomal damage at <10µM. On the other hand, GA prevented H2O2-induced chromosomal damage in a dose-dependent manner with a significant effect detected at 1µM. Chromosomal damage induced by tert-butylhydroperoxide was not prevented by GA even at 10µM. These results suggest that physiological concentration of tea constituents are not genotoxic but rather anti-genotoxic against ROS, although their preventive effects are slightly different depending on their chemical structure.

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Since the frequency of spontaneous mutation in bacteria, however, is extremely low, generally 10−8–10−10, it is difficult to detect suppressive ability on spontaneous mutation significantly.It has been known that mutT in Escherichia coli is the gene which cleans up 8-oxo-dGTP as oxidative damage in nucleotide pools. Since the mutant deficient in mutT shows very high frequency of spontaneous mutation, roughly 10−5–10−6, it may be useful to examine suppressive ability of the substances on spontaneous mutation of the mutant deficient in mutT. For examination of suppressive ability of the substances on spontaneous mutation, it is necessary to start incubation for the test from sufficiently low numbers of cells because of the high frequency of spontaneous mutation in mutT strain. For this purpose, it was considered that the technique of the Luria & Delbruck fluctuation test [8] using 96-well microtiter plate for detecting environmental mutagens is applicable. The present paper describes a new method, called Mut-Test, developed by us, applying the technique of the fluctuation test, to examine suppressive abilities of two hydroxyl radical scavengers: d(−)-mannitol and thiourea, which were used as the positive controls and to detect those of four vitamins: l-ascorbic acid, b-carotene, folic acid and riboflavin, and 10 polyphenols: caffeic acid, ellagic acid, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, gallic acid, pyrocatechol, pyrogallol, quercetin and tannic acid which are recognized as antimutagens, in E. coli WP2mutT mutant with high spontaneous mutation frequency due to oxidative damage. Furthermore, the concentrations for 50% of suppressive ability in five positive samples are compared.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Gallic acid purchased from nacalai tesque (Kyoto, Japan).

Target gene:

mutT gene

Species / strain / cell type:

E. coli WP2

Additional strain / cell type characteristics:

other: deficient in muT

Test concentrations with justification for top dose:

The negative sample of gallic acid and its highest concentration used: 5.88x10E-1

Vehicle / solvent:

Distilled water

Untreated negative controls:

yes

Remarks:

distilled water for gallic acid

Positive controls:

yes

Positive control substance:

other: distilled water and d(-)-mannitol

Details on test system and experimental conditions:

Construction of E. coli strain WP2 deficient in the mutT gene
The 0.9 kb PvuII–PvuII fragment of E. coli chromosome carrying mutT was isolated from lambda 15B8 of the Kohara library and cloned into the PvuII site of vector plasmid pUC19, giving pDU1. The 1.3 kb DNA fragment carrying the kanamycin resistance
(KmR) gene which was isolated from plasmid pUC4K was ligated with the pDU1 digested with EcoRI and HindIII, the site of which is located in the center of mutT. Since the resulting plasmid pDU2 carries the KmR gene in the center of mutT, the gene function of mutT was inactivated. The plasmid pDU2 was digested with ScaI and the resulting linear DNA fragment (about 4 kb) was purified. This fragment was introduced into strain JC7623 (recBC, sbcB). KmR transformants were selected. The KmR
marker was transferred into the WP2 strain (trpE65, malB15, lon-11, sulA1) by transduction with P1 bacteriophage. The resulting KmR colonies on the selective plate were randomly selected and their spontaneous mutation frequencies were estimated. The colonies, which showed high spontaneous mutation frequencies, were isolated as the WP2mutT strain and confirmed by southern hybridization analysis.

Mut-Test
An amount of 0.4 ml of the culture (4x10E7 to 5x10E7 cell/ml) of WP2mutT resuspended in M9 buffer was inoculated into 40 ml of Davis–Mingioli salts with addition of 800mg/ml of glucose, 0.25 mg/ml of tryptophan, 5mg/ml of bromocresol purple as an indicator
at the final concentration and 0.4 ml of the samples at the concentrations indicated in the tables and/or figures. For the control, 0.4 ml of distilled water or 0.4 ml of DMSO was used instead of the sample. 0.25 ml aliquots of the mixture were aseptically
dispensed in to 96-well microtitre plates by using Transtar-96 (Costar, Cambridge, USA). The number of cells per well at the starting time was approximately 5.0x10E2. After 48 h of incubation at 37°C, the numbers of the wells (trpC revertant-well) in which the culture became turbid and changed color from purple to yellow due to growth of spontaneous trpC revertant were counted. Growth and change of color in the cultures in the wells show that spontaneous mutation was not suppressed by the samples. The results were obtained by the average of the triplicate experiments. The numbers of trpC revertant-well of each positive sample and their concentrations were indicated in the vertical and horizontal axes, respectively.

Evaluation criteria:

Growth and change of color in the cultures in the wells show that spontaneous mutation was not suppressed by the samples.

Species / strain:

E. coli, other: muT

Metabolic activation:

not applicable

Genotoxicity:

other: negative antimutagenic effect

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

In Table 1, the numbers of viable cells, spontaneous trpC revertants per SEM plates and trpC revertants per 10E7 viable cells of WP2 and WP2mutT were shown. As shown in the table, the frequency of spontaneous mutation in WP2mutT was approximately 1000-fold of the wild type strain, WP2.
Suppressive abilities of the samples in their concentrations on spontaneous mutation were represented by the numbers of trpC revertant-well. The numbers of trpC revertant-well were 90±4 in the case of distilled water, and 9±2 and 8 ± 1 in the cases of d(−)-mannitol at 0.01mM and thiourea at 5 mM, respectively, as the positive controls.
Gallic acid showed negative in Mut-Test at the concentration of 5.88x10E-1 which is nearly saturated solution.

Table 1 : Spontaneous mutation of E.coli WP2mutT and its wild type strain^a

Strains	Number of colonies/SEM plate^b	Spontaneous mutant colonies/SEM plate	Trp^+revertants/10^7viable cells
WP2	47 ± 4c	9 ± 2	1.94
WP2mutT	38 ±5	7904 ± 28	2080

a The numbers represent the average of triplicate plates.

b Diluted to 10⁻⁶.

c Mean_range.

Conclusions:

d(−)-Mannitol and thiourea used as the positive controls have been known to be effective hydroxyl radical scavengers [18–20]. They showed clear suppressive ability, indicating that they scavenge hydroxyl radicals and suppress oxidative damage such as 8-oxo-dGTP in nucleotide pools and suggesting that Mut-Test is useful for the samples. Gallic acid showed negative in Mut-Test to mutagenesis. Gallic acid which has been reported to be antimutagenic to mutagenesis of UV, Trp-P-1, etc. showed negative up to the concentration of 5.88x10E-1. It may be possible to consider that all negative samples in Mut-Test do not suppress spontaneous mutation due to oxidative damage such as 8-oxo-dGTP in nucleotide pools, suggesting that their antimutagenic effects on cells may not be related to oxidative damage in cells. It can be considered that the conditions of the samples such as molecular structure, chemical form and membrane permeability may be implicated in the different results between the positive and the negative samples. For instance, it has been reported recently that antimutagenic effects of some antioxidative polyphenols are related to affinities to lipid bilayers.

Executive summary:

Since it has been considered that suppression of spontaneous mutation in cells is related to suppression of spontaneous carcinogenesis, it is significant to detect substances which suppress spontaneous mutation in bacterial cells such as Escherichia coli and Salmonella typhimurium in the environment. However, since the frequency of spontaneous mutation in bacteria is usually very low, generally 10E−8–10E−10, it is difficult to determine significant suppressive ability of such substances on spontaneous mutation. A new method, Mut-Test, was developed by us, applying Luria & Delbruck fluctuation test, to detect substances which suppress spontaneous mutation using E. coli mutT mutant in which spontaneous mutation frequency due to oxidative damage is enhanced to approximately 500–1000 times of the wild type strain. Suppressive abilities of two hydroxyl radical scavengers: d(−)-mannitol and thiourea, were examined and clear positive results were obtained, suggesting that the radical scavengers are suitable as the positive control for the test. Using Mut-Test, suppressive abilities of four vitamins: l-ascorbic acid, b-carotene, folic acid and riboflavin; 10 polyphenols: caffeic acid, ellagic acid, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, gallic acid, pyrocatechol, pyrogallol, quercetin and tannic acid which are recognized as antimutagens, were examined. Furthermore, the concentrations for 50% of suppressive abilities of five positive samples, l-ascorbic acid, folic acid, caffeic acid, pyrocatechol and pyrogallol were compared. Negative results were obtained in nine samples, riboflavin, tannic acid, etc. suggesting that their antimutagenic effect on cells may not be related to oxidative damage in cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)