Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of sperimental phase:01June 2017; End of experimental phase: 26 June 2017; Study completion: 25 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Target gene:
the test item for the ability to induce gene mutations in Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these
stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures,
which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement No Growth onMinimal plates+Biotin.Growth onMinimal plates+Biotin+Histidine.
Tryptophan requirement No Growth onMinimal agar plates.Growth onMinimal plates+Tryptophan.
-uvrA, uvrB : Sensitivity to UV irradiation.
-rfa : Sensitivity to Crystal Violet.
- pKM101: Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity
Metabolic activation:
with and without
Metabolic activation system:
liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone in Main Assay I and liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification), in Main Assay II.
Test concentrations with justification for top dose:
Preliminary Toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate
Main Assay I: +/- S9: 5000, 2500, 1250, 625 and 313 µg/plate
Main Assay II: +/- S9: 5000, 2500, 1250, 625 and 313 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
methylmethanesulfonate
other: 2-aminoanthracene, Trypan blue Solution 0.4%
Remarks:
Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
Details on test system and experimental conditions:
The preliminary toxicity test and the first experiment were perfomed using a plate-incorporation method. The second experiment was performed using a pre-incubation method.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Doubling rate ( Chu et al. 1981); Regression line

Results and discussion

Test results
Key result
Species / strain:
other: S.typhimurium TA1535, TA1537, TA98 and TA100; E.coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item did not induce relevant increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item Reactive Orange 12 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism,under the reported experimental conditions.

Executive summary:

The test item Reactive Orange 12 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone (standard metabolic activation) inMain Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification), in Main Assay II. The test item was used as a solution in sterile water for injection.

Toxicity test

The test item Reactive Orange 12 was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Neither relevant toxicity, nor relevant increases in revertant numbers were observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

Main Assays

On the basis of the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate. At the end of the incubation period, no toxicity of the test item was observed, with any tester strain, at any dose level, in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested,Main Assay II was performed. Based on the chemical structure of the test item (azo-dyes), the experiment was performed using the pre-incubation method in the presence of a reductive metabolic system (hamster S9 supplemented with flavin mononucleotide cofactor). The test item was assayed at the same concentrations used inMain Assay I. Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. No precipitation of the test item was seen at the end of the incubation period, at any concentration tested, in the absence or presence of S9 metabolism, in any experiment. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system.

Conclusion

It is concluded that the test item Reactive Orange 12 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.