Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Eukesolar Rubine EB Liquid - dried
- Physical state: Solid / dark red
- Analytical purity:
HPLC fingerprint:
87.0 area-% (268 nm) sum of all peaks: 99.4 area-%
90.1 area-% (552 nm) sum of all peaks: 100.2 area-%
(For details see analytical report No.: 14L00142)
- Lot/batch No.: Dye powder sample 14/076 from Material no. 52631478; batch no. M-R/G
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Method

Target gene:
HIS /TRP
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (rat and hamster)
Test concentrations with justification for top dose:
33 μg - 5 200 μg/plate (SPT)
33 μg - 5 200μg/plate (Prival)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: DMSO was a suitable vehicle
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
congo red
other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was
based on the method of Ames et al.

Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL
agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino
acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept
in a water bath at about 42 - 45°C, and the remaining components were added in the
following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies
(his+ revertants) were counted. The colonies were counted using the Sorcerer Image
Analysis System with the software program Ames Study Manager (Perceptive Instruments
Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance
hinders the counting using the Image Analysis System.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL
agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino
acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water
bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies
(his+ revertants) were counted. The colonies were counted using the Sorcerer Image
Analysis System with the software program Ames Study Manager (Perceptive Instruments
Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance
hinders the counting using the Image Analysis System.

Prival Preincubation Test
The experimental procedure is based on the method described by Yahagi et al. and
Matsushima et al. and has been modified further to include reductive conditions by Prival
et al.
0.1 mL test solution or vehicle (negative control), 0.1 mL bacterial suspension and 0.5 mL S9
mix (with metabolic activation) or phosphate buffer (without metabolic activation) were
incubated at 30°C for 30 minutes using a shaker. Subsequently, 2 mL soft agar which
consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution
(minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM
biotin or 0.5 mM tryptophan) were added. After mixing, the samples were poured onto the
Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
5 g D-glucose, monohydrate
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted.
The colonies were counted using the Sorcerer Image Analysis System with the software
program Ames Study Manager (Perseptive Instruments Ltd., Haverhill, UK). Colonies were
counted manually, if precipitation of the test substance hindered the counting using the
Image Analysis System.


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Evaluation criteria:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or
above.
• Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and
E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA
1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either
without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control
range under all experimental conditions in at least two experiments carried out
independently of each other.
Statistics:
N/A

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Additional information on results:
TOXICITY
A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was occasionally
observed in the standard plate test depending on the strain and test conditions from about
1 000 μg/plate onward.
In the prival preincubation assay bacteriotoxicity (reduced his- background growth, decrease
in the number of his+ or trp+ revertants) was occasionally observed depending on the strain
and test conditions from about 1 000 μg/plate onward.

SOLUBILITY
Test substance precipitation was found from about 2 600 μg/plate onward with and without S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a relevant

increase in the number of revertant colonies either without S9 mix or after adding a

metabolizing system in two experiments carried out independently of each other (standard

plate test and prival preincubation assay).

Besides, the results of the negative as well as the positive controls performed in parallel

corroborated the validity of this study, since the values fulfilled the acceptance criteria of this

study.

In this study with and without S9 mix, the number of revertant colonies in the negative

controls was within or marginally below the range of the historical negative control data for

each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant

increase in the number of revertant colonies within the range of the historical positive control

data or above.

Applicant's summary and conclusion