1,5-bis(cyclohexylamino)anthraquinone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Data is from publication.

Data source

Reference

Reference Type:

publication

Title:

Effects of various plant polyphenols on bladder carcinogen benzidine-induced mutagenicity

Author:

Patrudu S. Makena *, King-Thom Chung

Year:

2007

Bibliographic source:

Food and Chemical Toxicology 45 (2007) 1899–1909

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

To evaluate the mutagenic potential of (-)-Epigallocatechin in Salmonella typhimurium TA102 by Bacterial reverse mutation assay.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

970-74-1

Details on test material:

- Name of test material (as cited in study report): (-)-Epigallocatechin
- Substance type: Organic

Specific details on test material used for the study:

Details on test material
- Name of test material (as cited in study report): (-)-Epigallocatechin
- Substance type: Organic

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

The rat liver enzyme S9 ,1254 Aroclor induced in 0.154 M KCl.

Test concentrations with justification for top dose:

0 and 50 µg/plate

Vehicle / solvent:

Vehicle
- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Benzidine

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: Preincubation Method
DURATION
- Preincubation period:20 min
- Exposure duration:48 hours

NUMBER OF REPLICATIONS: Triplicates
DETERMINATION OF CYTOTOXICITY
- Method: % Cell viability was observed.

Rationale for test conditions:

Not specified.

Evaluation criteria:

The histidine revertant colony numbers were counted and compare to positive and negative control.

Statistics:

All experiments were repeated for the reproducibility. The values are means of revertants/plate ± standard deviations (Using Microsoft Excel) of one or
two experiments, each with three plates/dose. The statistical significance was calculated using student T-test (using the TTEST program in Microsoft Excel
software:Microsoft, Redmond, WA), and all p values were less than 0.05.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: The experimental procedure follows standard trypan blue exclusion using hemocytometer. One hundred ll of overnight culture (10–12 h) of Salmonella TA102 was incubated at 37 C with 100 ll of each test chemical in the presence and absence of 500µl of 4% rat liver S9 mix. After 4 h of incubation, 50 ll of the mixture was mixed with 50µ of 0.04% trypan blue dye and 20 ll of this mixture was loaded on to the hemocytometer and counted the viable cells (unstained) and dead cells (stained) under microscope (40• magnification) to estimate cytotoxicity caused by the test chemicals. One hundred ll of overnight culture of Salmonella TA102 (without tested chemical) was stained for the viable cell count as a control and the value was taken as 100%. The percent viability was calculated
using the formula given below:

% Cell viability =Total viable cells (stained) /Total cells (stained plus unstained)X100

The Cytotoxicity test was negative for (-)-Epigallocatechin

Remarks on result:

other: No mutagenic effect were observed

Any other information on results incl. tables

Mutagenicity of plant polyphenols on Salmonella TA102 tester strain

Sr no.	Test chemicals (50µg/plate)	Number of revertants
		With S9	Without S9
1	Benzidine (positive control)	1022 ± 24	383 ± 55
2	DMSO (negative control)	311 ± 11	293 ± 9
3	(-)-Epigallocatechin	669 ± 37	608 ± 9

 Effect of various polyphenols such as flavonoids, stilbenes, and phenolic

acids on TA102 with and without S9 mix. The values are means of revertants/

plate ± standard deviations of one experiment, each with three

plates/dose.

Applicant's summary and conclusion

Conclusions:

(-)-Epigallocatechin was evaluated for its mutagenic potential in Salmonella typhimurium TA102 by Ames Salmonella/microsomal mutagenicity inhibition test. The test result was considered to be negative in the presence and absence of S9.

Executive summary:

(-)-Epigallocatechin was assessed for its possible mutagenic potential .For this purpose Ames Salmonella/microsomal mutagenicity inhibition test was performed on Salmonella typhimurium TA102. The test material was exposed at the concentration of 0 and 50 µg/plate in the presence and absence of mutagenic potential. No mutagenic effects were observed in the presence and absence of S9. Therefore (-)-Epigallocatechin was considered to be non mutagenic for Salmonella typhimurium TA102. Hence the test substance cannot be classified as genetox in vitro.