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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: reverse gene mutation assay with Salmonella typhimurium
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 November 2002 - 3 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate
IUPAC Name:
Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Aurantiol Pure
- Physical state: Yellow viscous liquid

- Expiration date of the lot/batch: February 02, 2015
Specific details on test material used for the study:
Batch No.: 9000474306
Purity: 98.4%
Aggregate State at Room Temperature: liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000 and 2500 µg/plate
Vehicle / solvent:
DMSO (MERCK, D- 64293 Darmstadt; purity > 99 % ). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
Standard Ames conditions.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
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
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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

Any other information on results incl. tables

DISCUSSION OF RESULTS

The test item Aurantiol Pure was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; and 2500 µg/plate

 

Toxic effects were observed at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

/

/

2500

2500

TA 1537

/

5000

1000, 2500

1000, 2500

TA 98

1000, 5000

5000

1000, 2500

/

TA 100

1000 - 5000

2500, 5000

100 - 2500

2500

TA 102

1000 - 5000

1000 - 5000

1000, 2500

2500

 

No visible reduction of the background growth was observed up to the highest concentration.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Aurantiol Pure at any concentration 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.

The historical range of positive controls was exceeded in strains TA 1535 (Exp. I) iwthout metabolic activation and in strain TA 102 (exp.II) with metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay. In experiment I, the data in the negative and solvent control of strain TA 100 did not quite reach the lower limit of the historical control range. Since this deviation is rather small, this effect os considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, AURANTIOL PURE is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of AURANTIOL PURE to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; and 2500 µg/plate

Toxic effects were observed at higher concentrations with and without metabolic activation in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with AURANTIOL PURE 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.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, AURANTIOL PURE is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.