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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Mutagenicity
OECD 471 Ames - Target 
AURANTIOL PURE is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
GHS EU: NA
GHS UN: NA

Mutagenicity
OECD 476 Mouse Lymphoma- Target 
It is concluded that Aurantiol Pure is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
GHS EU: NA
GHS UN: NA

Mutagenicity
OECD 487 Mirconucleus- Target 
it is concluded that this test is valid and that Aurantiol Pure is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
GHS EU: NA
GHS UN: NA
Link to relevant study records

Referenceopen allclose all

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
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
Specific details on test material used for the study:
Batch No.: 9000474306
Purity: 98.4%
Aggregate State at Room Temperature: liquid
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.
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.
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

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.

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
In cultured peripheral human lymphocytes
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 09 May 2014 and 25 June 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Batch No.: SC00010629
Description: Yellow viscous liquid
Purity/Composition: UVCB substance
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Not clastogenic or aneugenic in human lymphocytes.
Conclusions:
it is concluded that this test is valid and that Aurantiol Pure is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

Introduction

The study was conducted according to a method that was designed to evaluate Aurantiol Pure for its ability to induce micronuclei on cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9 -mix). The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals, Guideline No. 487: In Vitro Mammalian Cell Micronucleus Test (adopted 22 July 2010) and the European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.49 “In Vitro Mammalian Cell Micronucleus Test". Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

General Information

An in vitro micronucleus assay with Aurantiol Pure in cultured peripheral human lymphocytes (with independent repeat). This report describes the effect of Aurantiol Pure on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of Aurantiol Pure was tested in two independent experiments. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch SC00010629 of Aurantiol Pure was a yellow viscous liquid. Aurantiol Pure was dissolved in dimethyl sulfoxide.

Methods

In the first cytogenetic assay, Aurantiol Pure was tested up to 333 μg/ml for a 3 hour exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Aurantiol Pure precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Aurantiol Pure was tested up to 166 μg/ml for a 24 hour exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

Results

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition in the first experiment colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Aurantiol Pure did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Conclusion

Finally, it is concluded that this test is valid and that Aurantiol Pure is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: cell gene mutation with L5178Y mouse lymphoma cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 29 July 2014 and 20 October 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
Batch No.: SC00010629
Description: Yellow viscous liquid
Purity: UVCB
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
It is concluded that Aurantiol Pure is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Tests" adopted 21 July 1997 and Method B17: "Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test" of Commission Regulation (EC) No. 440/2008 of 31 May 2008.

Methods

Evaluation of the mutagenic activity of Aurantiol Pure in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat).

This report describes the effects of Aurantiol Pure on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch SC00010629 of Aurantiol Pure was a yellow viscous liquid. The test substance was dissolved in dimethyl sulfoxide.

Experiments

In the first experiment, Aurantiol Pure was tested up to concentrations of 270 μg/ml and 512 μg/ml in the absence and presence of 4% (v/v) S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 10 and 35% in the absence and presence of S9-mix, respectively. Aurantiol Pure precipitated in the exposure medium at dose levels of 175 μg/ml.

In the second experiment, Aurantiol Pure was tested up to concentrations of 20 and 512 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation times were 24 hours for incubations in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix. Relative total growth (RTG) was reduced to 3 and 72% in the absence and presence of S9-mix, respectively. Aurantiol Pure precipitated in the exposure medium at dose levels of 200 μg/ml.

Mutation frequencies in cultures treated with positive control chemicals were increased 7- and 11-fold for MMS in the absence of S9-mix, and 8-and 10-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. Although the mutation frequency of the positive control in the presence of S9-mix was not within the acceptability criteria, the mutagenic response was more than 8 times greater than the concurrent solvent control values and therefore the validity of the test was considered to be not affected.

Results

In the absence of S9-mix, Aurantiol Pure did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Aurantiol Pure did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

Conclusion

It is concluded that Aurantiol Pure is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification