Cyperus scariosus, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (similair to OECD 471): non-mutagenic with or without metabolic activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 03 2012- May 06 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The Ames microplate assays are modified fluctuation tests using 384-well plates (48 wells per sample, per dose).

The modified microplate test, Ames MPF PENTA I, in brief is as follows:
1. Pre-growth of tester strains overnight in Oxoid broth.
2. A 90 minute incubation in Exposure Medium with limiting histidine/tryptophan in the presence of test sample and S9 if employed.
3. Distribution and plating of cells in a medium which selects for revertants. This medium is free of histidine/tryptophan and contains a pH indicator dye that turns
from purple to yellow upon colony growth.
4. Incubation of the microtiter plates for 48 hours at 37C to allow growth of revertant colonies.
5. Scoring of microtiter plates for positive (yellow) wells, data entry, and evaluation of mutational potential.

GLP compliance:

no

Remarks:

The assays reported in this document were performed according to Xenometrix’ Quality Assurance SOP’s.

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Absense of S9: 50, 15.8, 5, 1.58, 0.5, 0.158 ppm. Cytotoxicity is used for justification for top dose.
Presense of S9: 2000, 633, 200, 63.3, 20, 6.3 ppm

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

other: 2-nitrofluorone, N-aminocytidine, 2-aminoantracene

Evaluation criteria:

The following criteria were used to evaluate the results: fold increase over the solvent control baseline, dose-dependency and the Student’s t-test. The fold increase of revertants relative to the solvent control was determined by dividing the mean number of positive wells at each dose by that of the solvent control baseline. The solvent control baseline was derived from the mean number of positive wells in the solvent control plus 1 standard deviation. If the baseline was below 1, it was set to 1. There were 3 solvent control replicates for each test condition (e.g. TA98 without S9, TA98 with S9 etc.). A fold increase equal to or greater than 2 times the baseline level (red-dotted line in graphs below) is rated as possibly positive in the assay. Multiple responses of greater than 2-fold the baseline level with a doseresponse will lead to the test compound being classified as a clear positive. A test compound is classified negative where no response greater than 2 times the baseline is recorded. Student’s t-test (unpaired, one-sided) has been used to determine significance at the a = 0.05 level. Although statistical analysis has been applied to all data collected, increases in revertant yields are not classified as positive if less than 2.0-fold over the baseline value, and if no dose-response is observed.

Statistics:

Student's t-test

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

In the main assay with all strains, in the absence of S9, there was visible and statistically significant cytotoxicity of test item in TA100 and TA1537 at the highest concentration (50 ppm). With S9, no sign of cytotoxicity in strains TA98 and EC Combo was observed. Indications of cytotoxic effect between 200 and 2000 ppm were observed in the other strains: in TA100 and TA1535, the three highest doses resulted in zero positive wells and in TA1537 in a decrease of the positive wells as compared to the solvent control. However, no increase in the intensity of the color of the medium was observed at these concentrations.

Conclusions:

Cyperus scariosus, ext. showed no increases of greater than 2-fold the baseline in any strain and under any conditions at the non-cytotoxic concentraions. According to the Ames MPF PENTA I data, Cyperus scariosus, ext. is negative for mutational activity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The objective of the study was to determine the potential of Cyperus scariosus, ext. and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The method used was Ames MPF PENTA I assay.

For the pre-screen assay, the test item was tested up to concentrations of 40 000 ppm in the strain TA100. The concentrations chosen were all cytotoxic when tested in the absence of S9, whereas with S9, no clear toxic effect observable.

In the second assay, the test item was tested in absence of S9 up to concentrations of 50 ppm and in the presence of S9 up to concentrations of 2000 ppm.

There was no increase the number of revertants greater than two-fold the baseline in any strains and under any condition at the non-cytotoxic doses. According to the criteria to evaluated MPR PENTA I data, test sample Cyperus scariosus, ext. is negative at the concentrations tested.

Justification for classification or non-classification

Based on the results of in vitro bacterial gene mutation study, no classification is proposed for genotoxicity according to the criteria of CLP regulation 1272/2008.