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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase:01 June 2017; End of experimental phase: 26 June 2017; Study completion: 25 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
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

Constituent 1
Chemical structure
Reference substance name:
Sodium bis[4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxy-N,N-dimethylbenzene-1-sulphonamidato(2-)]chromate(1-)
EC Number:
278-137-2
EC Name:
Sodium bis[4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxy-N,N-dimethylbenzene-1-sulphonamidato(2-)]chromate(1-)
Cas Number:
75214-69-6
Molecular formula:
C36H34CrN10NaO8S2
IUPAC Name:
hydroxylamine
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:

Tester strain S9 Dose level (µg/plate)

TA1535, TA98, WP2 uvrA ± 5000, 2500, 1250, 625, 313
TA1537 ± 5000, 2500, 1250, 625, 313, 156
TA100 − 2500, 1250, 625, 313, 156, 78.1
TA100 + 5000, 2500, 1250, 625, 313

Main Assay II: .

Tester strain S9 Dose level (µg/plate)

TA1535, TA98, WP2 uvrA ± 5000, 2500, 1250, 625, 313
TA1537 ± 5000, 2500, 1250, 625, 313, 156
TA100 − 625, 313, 156, 78.1, 39.1
TA100 + 5000, 2500, 1250, 625, 313

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:
cytotoxicity
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 Acid Red 279 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 Acid Red 279 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 dimethylsulfoxide (DMSO).

Toxicity test

The test item Acid Red 279 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. At the end of the incubation period, precipitation of the test item, evident to the unaided eye, was observed with all tester strains at the highest dose level, both in the absence and presence of S9 metabolism. The precipitate interfered with the evaluation of the background lawn but allowed the scoring of revertant numbers at this dose level. Relevant toxicity was seen with TA100 tester strain at the two highest dose levels in the absence of S9 metabolism. No increase in revertant numbers was 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, inMain Assay I, using the plate incorporation method and the standard metabolic activation system, the test item was assayed at the following dose levels:

Tester strain -S9-Dose level (µg/plate)

TA1535, TA98, WP2 uvrA ± 5000, 2500, 1250, 625, 313

TA1537 ± 5000, 2500, 1250, 625, 313, 156

TA100 − 2500, 1250, 625, 313, 156, 78.1

TA100 + 5000, 2500, 1250, 625, 313

Toxicity was observed in the absence of S9 metabolism with TA1537, TA98 and TA100 tester strains at higher dose levels. Precipitation, which interfered with the evalutation of the background lawn only, was seen at the highest dose level of 5000 µg/plate, both in the absence and presence of S9 metabolism.

Toxicity was observed in the absence of S9 metabolism with TA1537, TA98 and TA100 tester strains at higher dose levels. Precipitation, which interfered with the evalutation of the background lawn only, was seen at the highest dose level of 5000 µg/plate, both in the absence and 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 following dose levels:

Tester strain- S9- Dose level (µg/plate)

TA1535, TA98, WP2 uvrA ± 5000, 2500, 1250, 625, 313

TA1537 ± 5000, 2500, 1250, 625, 313, 156

TA100 - 625, 313, 156, 78.1, 39.1

TA100 + 5000, 2500, 1250, 625, 313

Slight signs of toxicity were observed at higher dose levels (5000, 2500 µg/plate) with TA1537 and TA98 tester strains in the absence of S9 metabolism and with WP2 uvrA and TA100 tester strains in the presence of S9 metabolic activation. A more pronounced toxic effect was noted with TA100 tester strain showing thinning of the background lawn and reduction in revertant numbers at 625 µg/plate. Precipitation of the test item was seen in both experiments at the highest or two highest dose levels in the absence and presence of S9 metabolism. Although the precipitate, evident to unaided eye, was mild, it interfered with the microscopic analysis of the background lawn at 5000 µg/plate.

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 Acid Red 279 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

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