Bulnesia sarmienti, ext., acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 July 2004 to 20 July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced with Phenobarbital / β-Naphtoflavone

Test concentrations with justification for top dose:

- Pre-experiment/Experiment I (Plate incorporation test):
TA 1535, TA 1537, TA 98, TA 100 and TA 102 (without and with S9): 3; 10; 33; 100; 333; 1000; 2500, and 5000 µg/plate
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I since only minor toxic effects were observed and 5000 µg/plate were chosen as maximal concentration. In addition the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

- Experiment II (Pre-incubation test):
TA 1535, TA 1537, TA 98, TA 100 and TA 102 (without and with S9): 33; 100; 333; 1000; 2500, and 5000 µg/plate.

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

(untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Pre-experiment / Experiment I:in agar (plate incorporation method)
- Experiment II (independent repeat): pre-incubation method

DURATION
- Preincubation period: 60 minutes (Experiment II, pre-incubation method)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- For each strain and dose level, including the controls three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial beckground lawn.

DATA RECORDING
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben).

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

PRE-EXPERIMENT / EXPERIMENT I:
- The pre-experiment is reported as experiment I, since only minor toxic effects were observed and 5000 µg/plate was chosen as maximal concentration and since the following criteria was met: Evaluable plates (> 0 colonies) at five concentrations or more in all strains used.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative, solvent and strain-specific positive control values were within the laboratory historical control data ranges except for the positive control for strain TA102 in experiment I, with and without metabolic activation. This control exceeded the historical range. This effect indicates the sensitivity of the strain rather than compromising the assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Contradicting statements are reported for reduced background growth observed or not, at 5000 µg/plate with metabolic activation in all strains used in experiment I. As these plates were evaluable, and this is the limit concentration, this does not influence the reliability of the results. For experiment II no reduction of the background growth was reported.
- Slight toxic effects, evident as a reduction in the number of revertants, were observed with metabolic activation at 5000 µg/plate in strain TA1537 in experiment I, and at 2500 and 5000 µg/plate in strain TA 102 in experiment II.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 (1997).

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. At first a plate incorporation assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. Each concentration, including controls, was tested in triplicate. The test substance was tested at 33, 100, 333, 1000, 2500 and 5000 µg/plate, in both experiments. Negative controls (untreated and solvent) and strain specific positive controls were included. No substantial increase in revertant colony numbers of any of the five tester strains was observed after treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). In conclusion, it can be stated that under the experimental conditions of this study, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. At first a plate incorporation assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. Each concentration, including controls, was tested in triplicate. The test substance was tested at 33, 100, 333, 1000, 2500 and 5000 µg/plate, in both experiments. Negative controls (untreated and solvent) and strain specific positive controls were included. No substantial increase in revertant colony numbers of any of the five tester strains was observed after treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). In conclusion, it can be stated that under the experimental conditions of this study, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Justification for classification or non-classification

Based on the results of the Ames test, the substance does not have to be classified for mutagenicity in accordance with EU CLP and its amendments (EC 1272/2008).