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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames, OECD 471, GLP study: Negative.

Mouse Lymphoma Assay, OECD 490, GLP study: Negative

In vitro Micronucleus Test, OECD 487, GLP study: Negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the results of the first mutation experiment, BOURGEONAL was tested up to concentrations of 512 and 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the Salmonella typhimurium and Escherichia coli strain, respectively.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Details on test system and experimental conditions:
According to OECD 471
Rationale for test conditions:
According to OECD 471
Evaluation criteria:
According to OECD 471
Statistics:
According to OECD 471
Key result
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
Key result
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
Key result
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
Key result
Species / strain:
S. typhimurium TA 100
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:
E. coli WP2 uvr A
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
Conclusions:
In conclusion, based on the results of this study it is concluded that BOURGEONAL is not
mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli
reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of BOURGEONAL 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 study procedures described in this report were based on the most recent OECD, EC and

METI guidelines.

Batch SC00021197 of BOURGEONAL was a pale yellow liquid with a purity of 97.2%. The

test item was dissolved in dimethyl sulfoxide.

In the first mutation assay, the test item was tested up to concentrations of 5000 μg/plate in

the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at this

dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of

the bacterial background lawn and/or the presence of microcolonies, was observed in all tester

strains in the absence and presence of S9-mix.

In the second mutation assay, the test item was tested up to concentrations of 512 and

5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the Salmonella

typhimurium and Escherichia coli strain, respectively. The test item precipitated on the plates

at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of

revertants, reduction of the bacterial background lawn and or the presence of microcolonies,

was observed in all tester strains in the absence and presence of S9-mix. Except for tester

strain WP2uvrA in the presence of S9-mix where no toxicity was observed.

BOURGEONAL did not induce a significant dose-related increase in the number of revertant

(His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in

the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and

presence of S9-metabolic activation. These results were confirmed in an independently

repeated experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive

control items indicating that the test conditions were adequate and that the metabolic

activation system functioned properly.

In conclusion, based on the results of this study it is concluded that BOURGEONAL is not

mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli

reverse mutation assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes:
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
In the first cytogenetic assay, BOURGEONAL was tested up to 51 µg/mL and 60 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of
S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.
In the second cytogenetic assay, BOURGEONAL was tested up to 50 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and experimental conditions:
According to OECD guideline
Rationale for test conditions:
According to OECD guideline
Evaluation criteria:
According to OECD guideline
Statistics:
According to OECD guideline
Key result
Species / strain:
lymphocytes: lymphocytes
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
Conclusions:
In conclusion, this test is valid and BOURGEONAL is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate BOURGEONAL for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of BOURGEONAL was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

BOURGEONAL was a pale yellow liquid.The vehicle of the test item was dimethyl sulfoxide.

In the first cytogenetic assay, BOURGEONAL was tested up to 51 µg/mL and 60 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of
S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, BOURGEONAL was tested up to 50 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. 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, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system
(S9-mix) functioned properly.

BOURGEONAL did not induce a statistically significant and 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 experiments.

In conclusion, this test is valid and BOURGEONAL 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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
thymidine kinase (TK) locus in L5178Y
mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First Mutagenicity Test
Based on the results of the dose-range finding test, the following dose-ranges were selected
for the first mutagenicity test:
Without S9-mix: 0.31, 0.63, 1.25, 2.5, 5, 10, 15, 20, 25, 30 and 35 μg/ml exposure medium.
With S9-mix: 1.88, 3.75, 7.5, 15, 30, 40, 45, 50, 55, 60 and 65 μg/ml exposure medium.
Evaluation of toxicity
In the absence of S9-mix, the dose levels of 0.31 to 20 μg/ml showed no cytotoxicity.
Therefore, the dose levels of 0.63, 5 and 10 μg/ml were not regarded relevant for mutation
frequency measurement.
In the presence of S9-mix, the dose levels of 1.88 to 45 μg/ml showed no cytotoxicity.
Therefore, the dose levels of 3.75, 7.5 and 30 μg/ml were not regarded relevant for mutation
frequency measurement.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.31, 1.25, 2.5, 15, 20, 25, 30 and 35 μg/ml exposure medium.
With S9-mix: 1.88, 15, 40, 45, 50, 55, 60 and 65 μg/ml exposure medium.

Second Mutagenicity Test
To obtain more information about the possible mutagenicity of BOURGEONAL, a second
mutation experiment was performed in the absence of S9-mix with a 24 hour treatment
period.
Based on the results of the additional dose-range finding test, the following dose levels were
selected for mutagenicity testing 0.63, 1.25, 2.5, 5, 10, 15, 17.5, 20, 22.5, 25 and 30 μg/ml
exposure medium.
Evaluation of toxicity
The dose levels of 22.5 to 30 μg/ml were not used for mutation frequency measurement, since
these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were: 0.63, 1.25,
2.5, 5, 10, 15, 17.5 and 20 μg/ml exposure medium.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Accoridng to OECD guidelines
Rationale for test conditions:
Accoridng to OECD guidelines
Evaluation criteria:
Accoridng to OECD guidelines
Statistics:
Accoridng to OECD guidelines
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:
valid
Positive controls validity:
valid
Conclusions:
In conclusion, BOURGEONAL is not mutagenic in the mouse lymphoma L5178Y test
system under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the mutagenic potential of BOURGEONAL by

testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y

mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix).

The TK mutational system detects base pair mutations, frame shift mutations and small

deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in

the presence of S9-mix with a 3 hour treatment period.

The study procedures described in this report were based on the most recent OECD guideline.

Batch SC00021197 of BOURGEONAL was a pale yellow liquid. The test item was

dissolved in dimethyl sulfoxide.

In the first experiment, BOURGEONAL was tested up to concentrations of 35 μg/ml and

65 μg/ml in the absence and presence S9-mix, respectively. The incubation time was 3 hours.

Relative total growth (RTG) was reduced to 16% and 3% in the absence and presence of

S9-mix, respectively. The test item did not precipitate in the culture medium up to the dose

level of 65 μg/ml.

In the second experiment, BOURGEONAL was tested up to concentrations of 20 μg/ml in the

absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 10%. The

test item did not precipitate in the culture medium up to the dose level of 20 μg/ml.

The mutation frequency found in the solvent control cultures was within the acceptability

criteria of this assay

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced

significant increases in the mutation frequency. In addition, the mutation frequency found in

the positive control cultures was within the 95% control limits of the distribution of the

historical positive control database. It was therefore concluded that the test conditions were

adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, BOURGEONAL did not induce a significant increase in the

mutation frequency in the first experiment. This result was confirmed in an independent

experiment with modification in the duration of treatment.

In the presence of S9-mix, BOURGEONAL did not induce a significant increase in the

mutation frequency.

In conclusion, BOURGEONAL 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)

Genetic toxicity in vivo

Description of key information

No in vivo data was available and based on the in vitro data set available, no need for in vivo testing was deemed necessary.

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

Additional information

Justification for classification or non-classification

Based on the data available on Bourgeonal, no classification for genotoxicity is necessary according to the (EC) No 1272/2008 Regulation (CLP).