Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06.09.2018 to 22.10.2018
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: modified method by Prival et al. (1984) for azo dyes
Version / remarks:
1984
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
histidine operon
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:
Hamster S9
Test concentrations with justification for top dose:
0.35 - 5.63 mg/plate
Vehicle / solvent:
yes
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
cumene hydroperoxide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
The test substance is preincubated with the tester strain and sterile buffer (for
volume compensation) or the metabolic activation system for 30 minutes +/- 5 min. at 30°C
+/- 3°C. Subsequently the overlay agar is added and the mixture is poured on the surface of a
minimal agar plate. In addition deviating from the standard method a reductive metabolic
activation system with the reductive agent Flavinemononucleotide and with 30% uninduced
hamster S9 is applied.
Evaluation criteria:
The colonies were counted manually with a colony counter (Heathro Scientific LLC). The
mean value and standard deviation of the three resp. five replicates were calculated
(Microsoft Excel®). The resulting induction rates (IR = number of revertant colonies in the
plate with test substance / mean number of revertant colonies in the negative control plate
[NC]) were calculated for each test concentration and each tester strain.
Cytotoxic effects of the test item can be determined if the growth of the micro colonies in the
background lawn deviates from the negative control plates. A decrease in micro colonies
density or gaps in the bacterial background lawn are indicators for cytotoxic effects. These
are discovered by microscopic evaluation of the plates. Often such effects can be observed
in combination with reduced number of revertant colonies in comparison to the number of
spontaneous revertant colonies on the negative control plates (induction rate < 1). Toxic
effects to the bacteria are reported.
According to OECD Guideline 471 a sample is mutagenic if there is a concentration-related
increase over the range tested and/or a reproducible increase at one or more concentrations
in the number of revertant colonies per plate in at least one strain with or without the
metabolic activation system. Statistical methods may be used as an aid in evaluating the test
results. However, statistical significance should not be the only determining factor for a
positive result. For the evaluation of the results of the tests performed in this study the increase of revertants
in a sample is expressed as the induction rate [IR], (IR = number of revertants in the
sample/number of revertants in the control). In the case of a reproducible increase of the
number of revertants and if additionally a statistically significant difference between the mean
values can be demonstrated for example with the U-test according to Mann & Whitney e.g.,
the sample is evaluated as positive.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Red STR 172 is not mutagenic in the Ames Test modified by Prival.
Executive summary:

In the study five concentrations between 5.63 mg/plate and 0.35 mg/plate of the test item

“Red STR 172” corresponding with 5 mg/plate to 0.31 mg/plate of the active test substance

were applied. The test was performed with and without reductive metabolic activation with

pre-incubation and non-induced Hamster S9. Two independent studies were carried out.

In none of the tester strains a dose dependent increase of revertant colonies and no

reproducible increase at one or more concentrations in the number of revertant colonies per

plate with or without the metabolic activation occurred. The micro colonies in the bacterial

background lawn was not affected on any plate.

The results of the first and the second study (independent repetition) did not vary.