Registration Dossier

Administrative data

in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Form May 4 to June 2, 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
according to guideline
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Raction product of 2-Ethyl-1-hexylamine and 3-(2-Ethylhexyloxy)propylamine with 4-bromo-1,8 naphthalic anhydride
EC Number:
Cas Number:
Molecular formula:
Not applicable (UVCB substance)
Raction product of 2-Ethyl-1-hexylamine and 3-(2-Ethylhexyloxy)propylamine with 4-bromo-1,8 naphthalic anhydride
Test material form:
liquid: viscous
Specific details on test material used for the study:
- Name: YELLOW 2747
- Chemical Name: 1H-Benz[de]isoquinoline-1,3(2H)-dione, 6-amino-, N,N’bis
[mixed 2-ethylhexyl and 3-[(2-ethylhexyl)oxy]propyl] derivs
- Lot No.of test material: 78-242-15
- Expiration date of the lot: 01/02/2018

- Storage condition of test material: room temperature, protected from light


Species / strain
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:
S9 mix
Test concentrations with justification for top dose:
In two independent experiments several concentrations of the test item were used.
The concentrations, including the controls, were
tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Vehicle / solvent:
The test item was dissolved in DMSO and diluted prior to treatment.
Untreated negative controls:
Negative solvent / vehicle controls:
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
Five strains of S. typhimurium with the following characteristics were used:
TA 98:
his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA 100:
his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA 1535:
his G 46; rfa-; uvrB-: base-pair substitutions
TA 1537:
his C 3076; rfa-; uvrB-: frame shift mutations
TA 102:
his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions
Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA.
Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. They were
stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx.
8% v/v) over liquid nitrogen. All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for
histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the
polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell
permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a
protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting
many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes
(bacteria require biotin for growth).
The tester strains TA 98, TA 100 and TA 102 contain the R-factor plasmid, pkM101. These strains
are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor
parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an errorprone
DNA repair system which is normally present in these organisms
The properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and
tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly
according to Ames et al. . In this way it is ensured that the experimental conditions set up by
Ames are fulfilled.

Preparation of Bacteria
Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late
exponential or early stationary phase of growth (approx. 109 cells/mL). The nutrient medium consists
per litre:
8 g Nutrient Broth
5 g NaCl
A solution of 125 μL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the
phenotypic characteristics of the strain.

Agar Plates
The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames Test were prepared by
Eurofins Munich. Quality controls were performed.
Vogel-Bonner-salts contain per litre:
10 g MgSO4 x 7 H2O
100 g citric acid
175 g NaNH4HPO4 x 4 H2O
500 g K2HPO4
Sterilisation was performed for 20 min at 121 °C in an autoclave.
Vogel-Bonner Medium E agar plates contain per litre:
15 g Agar Agar
20 mL Vogel-Bonner salts
50 mL glucose-solution (40%)
Sterilisation was performed for 20 min at 121 °C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
7.0 g Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H2O
12.2 mg biotin
Sterilisation was performed for 20 min at 121 °C in an autoclave.
Rationale for test conditions:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean
values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.the biological relevance of the results is the criterion for the
interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible
biologically relevant positive response at any of the dose groups is considered to be non-mutagenic
in this system.
Evaluation criteria:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges
(mean values of the spontaneous reversion frequency are within the historical control data range
(2014 -2016)):
- S9 + S9
min max min max
TA 98 11 58 15 59
TA 100 49 155 62 160
TA 1535 4 41 3 38
TA 1537 3 35 3 36
TA 102 141 472 157 586
- corresponding background growth on negative control, solvent control and test plates is
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: all tester strains
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Exp. II: TA 100, TA 1535 at 5000 μg/plate (without S9mix). TA 1537 at 2500 μg/plate and higher (without S9mix).
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I. The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in tester strain TA 1537 at a concentration of 2500 μg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

In experiment II toxic effects of the test item were noted at concentrations of 2500 μg/plate and higher (without metabolic activation) depending on the particular tester strain.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with YELLOW 2747 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met.

Applicant's summary and conclusion

It can be stated that during the described mutagenicity test and under the experimental conditions reported, YELLOW 2747 did not cause gene mutations by base pair
changes or frameshifts in the genome of the tester strains used.
Therefore, YELLOW 2747 is considered to be non-mutagenic in this bacterial reverse mutation assay.