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 experimental phase: 03 August 2017;End of experimental phase 18 September 2017; Study completion: 05 February 2018
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:
2018
Report Date:
2018

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 (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

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.
Test concentrations with justification for top dose:
Preliminary Toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate

Main Assay I:

TA1535 and WP2 uvrA ± S9 : 200, 100, 50.0, 25.0, 12.5 and 6.25 µg/plate
TA98 ± S9 : 400, 200, 100, 50.0, 25.0 and 12.5 µg/plate
TA1537 ± S9 : 16.0, 8.00, 4.00, 2.00, 1.00 and 0.500 µg/plate
TA100 - S9 : 16.0, 8.00, 4.00, 2.00, 1.00 and 0.500 µg/plate
TA100 +S9 : 32.0, 16.0, 8.00, 4.00, 2.00, 1.00 and 0.500 µg/plate




Main Assay II:

TA1535 ± S9 : 80.0, 40.0, 20.0, 10.0, 5.00 and 2.50 µg/plate
TA1537 − S9 :10.0, 5.00, 2.50, 1.25 and 0.625 µg/plate
TA100 + S9 : 10.0, 5.00, 2.50, 1.25 and 0.625 µg/plate
TA1537 + S9 : 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/plate
TA100 - S9 : 10.0, 5.00, 2.50, 1.25, 0.625 and 0.313 µg/plate
WP2 uvrA ± S9 :160, 80.0, 40.0, 20.0, 10.0 and 5.00 µg/plate
TA98 ± S9 :320, 160, 80.0, 40.0, 20.0 and 10.0 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
methylmethanesulfonate
other: 2-aminoanthracene
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
Remarks:
For both Main assays toxicity, from slight to severe, was observed at the highest or at higher dose levels with all tester strains, both in the absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item did not induce two-fold 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 Basic Orange 22 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 Basic Orange 22 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.The test item was used as a solution in dimethylsulfoxide (DMSO).

Toxicity test

The test item Basic Orange 22 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.

Dose related toxicity, from severe to mild, was observed at all or almost all dose levels with all tester strains both in the absence and presence of S9 metabolic activation. A more pronounced toxic effect was observed with TA100 and TA1537 tester strains, showing severe toxicity at all concentrations tested. Based on these results, it was necessary to perform an additional toxicity test where the dose levels of 50.0, 15.8, 5.00, 1.58 and 0.500 µg/plate were assayed. Dose related toxicity was observed at the three highest dose levels both in the absence and presence of S9 metabolism. No remarkable increase in revertant colonies was noted with any tester strain/activation condition combinations. No precipitation of the test item was observed at the end of the incubation period at any dose level, in the absence or presence of S9 metabolism.

Main Assays

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method,the test item was assayed at the following dose levels:

Tester strain S9 Dose level (µg/plate)

TA1535 and WP2 uvrA ± S9: 200, 100, 50.0, 25.0, 12.5 and 6.25 (µg/plate)

TA98 ± S9: 400, 200, 100, 50.0, 25.0 and 12.5 (µg/plate)

TA1537 ± S9: 16.0, 8.00, 4.00, 2.00, 1.00 and 0.500 (µg/plate)

TA100 - S9: 16.0, 8.00, 4.00, 2.00, 1.00 and 0.500 (µg/plate)

TA100 + S9: 32.0, 16.0, 8.00, 4.00, 2.00, 1.00 and 0.500 (µg/plate)

Toxicity, as indicated by microcolony formation, thinning of the background lawn and/or reduction in revertant colonies was observed at higher concentrations with all tester strains both in the absence and presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested,Main Assay II was performed including a pre-incubation step for all treatments.

The concentration-range was slightly modified to take into account the toxicity observed in Main Assay I and the following dose levels were used:

TA1535 ± S9: 80.0, 40.0, 20.0, 10.0, 5.00 and 2.50 µg/plate

TA1537 − S9: 10.0, 5.00, 2.50, 1.25 and 0.625 µg/plate

TA100 + S9: 10.0, 5.00, 2.50, 1.25 and 0.625 µg/plate

TA1537 + S9: 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/plate

TA100 - S9: 10.0, 5.00, 2.50, 1.25, 0.625 and 0.313 µg/plate

WP2 uvrA ± S9: 160, 80.0, 40.0, 20.0, 10.0 and 5.00 µg/plate

TA98 ± S9: 320, 160, 80.0, 40.0, 20.0 and 10.0 µg/plate

Toxicity was observed at highest dose levels with all tester strains; a more pronounced toxic effect was generally observed in the absence of S9 metabolic activation.

No precipitation of the test item was observed in any experiment, at any dose level in the absence or presence of S9 metabolic activation.

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 S9 metabolism.

Conclusion

It is concluded that the test item Basic Orange 22 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.