Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

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

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

The antimutagenic activity of the major flavonoids of rooibos (Aspalathus linearis): Some dose–response effects on mutagen activation–flavonoid interactions

Author:

Snijman PW

Year:

2007

Bibliographic source:

Mutation Research 631 (2007) 111–123

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:

According to Ames et al. (Mutation Res., 31 (1975) 347-364)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Rutoside

EC Number:

205-814-1

EC Name:

Rutoside

Cas Number:

153-18-4

Molecular formula:

C27H30O16

IUPAC Name:

2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranoside

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: rutin was provided by Sigma-Aldrich.
- Purity, including information on contaminants, isomers, etc.: >95% pure;

Method

Target gene:

Histidine dependent gene.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Remarks:

Obtained from Prof. B.N. Ames, Berkeley University, CA, USA.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

Obtained from Prof. B.N. Ames, Berkeley University, CA, USA.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
Aroclor 1254-induced S9 homogenate (0.7 nmole cytochrome P450/mg protein), prepared from male Fischer rats, and incorporated in the S9 mixture at a level of 2mg protein/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of the test item

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: aflatoxin B1

Details on test system and experimental conditions:

MAIN STUDY:
The mutagenicity assay consisted of the addition of an overnight bacterial culture (100 µl), the diagnostic mutagen (100 µl), the test item (100 µl, concentration range, 0.001–1.2mM per plate) and 500 µl of S9 mix (2 mg protein per plate) to 2ml of top agar at 45ºC. The mixture was vortexed, poured onto a minimal glucose plate and incubated at 37ºC for 48 h in the dark. Five repetitions for each concentration were included. All experiments were repeated at least once and yielded similar data trends.

Evaluation criteria:

When the mutagenic response of the diagnostic mutagen was enhanced, the effect of the flavonoid as potential mutagen or promutagen was evaluated in the absence of the diagnostic mutagen with and without metabolic activation. The percentage inhibition/stimulation of the mutagen-induced response by the flavonoid was calculated using the formula:
{100−((Rs −R0)/(Rp −R0))×100}, where Rs is the average number of His+ revertants induced in the presence of the flavonoid, R0 the average number of His+ revertants in the absence of the mutagen (spontaneous revertants) and Rp is the average number of His+ revertants induced by the mutagen.

Statistics:

All individual groups were independent and tested for normality using the Kolmogorov–Smirnof test. Levene’s test was used to determine whether the groups had equal variances. Significant group differences were determined by the F-test (equality of variances) or the Welch test (inequality of variances), while the post hoc Tukey test was used to determine which groups differed significantly. The Student’s two-sample t-test was used to test for group differences when there were only two groups, with the Pooled method for groups with equal variances or the Satterthwaite method for groups with unequal variances. P < 0.05 indicated significant group differences.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
The test item reduced the number of 2-AAF-induced revertants in a typical dose response, showing a higher protective potency at lower concentrations. They exhibited similar antimutagenic responses against 2-AAF-induced mutagenesis at 0.4 and 0.08 mM, but stimulated the mutagenic response (P < 0.05) at the highest concentration (0.8 mM). Rutin exhibited a biphasic antimutagenic response against AFB1. The test item was found to be non-mutagenic inTA98 with/without the mutagen and/or metabolic activation.

Any other information on results incl. tables

Table 1. Percentage inhibition (−) or stimulation (+) of the mutagenic response of 2-AAF by test item (mM/plate) against tester strain S. typhimurium TA98 in the presence of metabolic activation.

Flavonoid (mM)	% inhibition(-)/stimulation(+) of mutagenic response
	1.2	0.8	0.6	0.4	0.12	0.08	0.06	0.04	0.01	0.008	0.006
Test Item	-	(+)14±4ghi^1	(−)0±4^2	(−)8±2efg^3	-	(−)11±4fgh^4	(−)17±6^5	-	-	-	(−)47±7^6

Values are the means±S.D. of five plates per treatment. Values in columns followed by the same letter do not differ significantly (P > 0.05); if letters differ, then P < 0.05. Values in rows followed by the same superscript number do not differ significantly (P > 0.05); if numbers differ, then P < 0.05. Average spontaneous revertants (n = 40) are 35±6; 2-AAF (5μg/plate) positive control 397±33.
^a Used as flavonoid reference. Spontaneous revertants subtracted prior to calculating the % inhibition.

 

Table 2. Percentage inhibition (−) or stimulation (+) of the mutagenic response of AFB1 (20 ng/plate) by test item (mM/plate) against tester strain S. typhimurium TA100 in the presence of metabolic activation.

Flavonoid (mM)	% inhibition(-)/stimulation(+) of mutagenic response
	1.2	0.8	0.6	0.4	0.12	0.08	0.06	0.04	0.01	0.008	0.006
Test Item	-	(-)47±3efg^1	(−)46±71	(−)48±9bcdefgh^1,2,3	-	(−)14±4ghij^2,3,4	(−)27±9^3	(−)44±12^1,2,3	-	-	(−)36±10^1,2

Values are the means±S.D. of five plates per treatment. Values in columns followed by the same letter do not differ significantly (P > 0.05); if letters differ, then P < 0.05. Values in rows followed by the same superscript number do not differ significantly (P > 0.05); if numbers differ, then P < 0.05. Average spontaneous revertants (n = 40) are 141±15; AFB1 positive control 416±38.

^a Used as flavonoid reference. Spontaneous revertants subtracted prior to calculating the % inhibition.
^b Cytotoxic effect.

 

Table 3. Mutagenic activity of selected flavonoids (mM/plate) exhibiting a comutagenic effect on 2-AAF (5μg/plate)-induced mutagenesis

Flavonoid (mM)	2-AAF	S9	Number of revertants
			1.2	0.8	0.6	0.4	0.12	0.08	0.06	0.04	0.01	0.008	0.006	0.001
Test item	+	+	-	388 ± 14^1	368 ± 14^2	310 ± 29^3	-	308 ± 18^4	305 ± 22^5	-	-	-	197 ± 27^6	-
	-	+	-	-	38 ±2	-	-	-	38 ±4	-	-	-	35 ±4	-
	-	-	-	-	37 ±3	-	-	-	36 ± 5	-	-	-	-	-

Values are the means±S.D. of five plates per treatment. Values in rows followed by the same superscript number do not differ significantly (P > 0.05); if numbers differ, then P < 0.05. Average spontaneous revertants (n = 40) are 35±6; 2-AAF positive control (+S-9) yielded 397±33 revertants; spontaneous revertants not subtracted from response.

Applicant's summary and conclusion

Conclusions:

The test item was found to be non-mutagenic in TA98 and TA100 with/without the mutagen and/or metabolic activation.

Executive summary:

A bacterial reverse mutation test was conducted on the test substance following a similar guideline to OECD's 471. The study was performed in S. typhimurium strains TA98 and TA100 in the absence and presence of metabolic activation. The test item was found soluble in DMSO which was used as vehicle. The metabolic activation system (S9 fraction) was provided by Aroclor 1254-induced S9 homogenate (0.7 nmole cytochrome P450/mg protein), prepared from male Fischer rats, and incorporated in the S9 mixture at a level of 2mg protein/ml. The mutagenicity assay consisted of the addition of an overnight bacterial culture (100 µl), the diagnostic mutagen (100 µl), the test item (100 µl, concentration range, 0.001–1.2mM per plate) and 500 µl of S9 mix (2 mg protein per plate) to 2ml of top agar at 45ºC. The mixture was vortexed, poured onto a minimal glucose plate and incubated at 37ºC for 48 h in the dark. Five repetitions for each concentration were included. All experiments were repeated at least once and yielded similar data trends.

It was found that he test item reduced the number of 2-AAF-induced revertants in a typical dose response, showing a higher protective potency at lower concentrations. They exhibited similar antimutagenic responses against 2-AAF-induced mutagenesis at 0.4 and 0.08 mM, but stimulated the mutagenic response (P < 0.05) at the highest concentration (0.8 mM). Rutin exhibited a biphasic antimutagenic response against AFB1. The test item was found to be non-mutagenic in TA98 and TA100 with/without the mutagen and/or metabolic activation.