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Toxicological information

Genetic toxicity: in vitro

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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: 04 July 2017; End of experimental phase: 31 July 2017; Study completion: 06 November 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 1997
Deviations:
yes
Remarks:
- Test item formulation in the preliminary toxicity test was carried out without correction for the test item displacement - In the Preincubation method, the test item was assayed at a maximum concentration of 10000 µg/plate, instead of 5000 µg/plate
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Target gene:
The test item 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.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
The E.coli used for this study was the strain WP2 uvrA.
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate 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, WP2 uvrA, TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate
- TA1537: ± S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate
- TA100 : ± S9: 2500, 1250, 625, 313 ,156 and 78.1 µg/plate

Main Assay II:
- TA1535, WP2 uvrA: ± S9: 10000, 5000, 2500, 1250 and 625 µg/plate
- TA100: :+ S9: 10000, 5000, 2500, 1250 and 625 µg/plate
- TA1537, TA100: - S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate
- TA1537: +S9: 10000, 5000, 2500, 1250, 625 and 313 µg/plate
- TA98: ± S9: 10000, 5000, 2500, 1250, 625 and 313 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water for injection.
- 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:
no
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 performed 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.typhyimurium 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:
not applicable
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, in the plate incorporation or pre-incubation assay, 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 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 HH-2015-623 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 sterile water for injection.

Toxicity test

The test item HH-2015-623 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 tested in the absence or presence of S9 metabolic activation. Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies, was observed with TA1537 and TA100 tester strains at the highest or two highest dose levels both in the absence and presence of S9 metabolic activation. No increase in revertant numbers was observed at any concentration tested with any tester strain/activation condition combinations.

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:

- TA1535, WP2 uvrA, TA98: ± S9:  5000, 2500, 1250, 625 and 313 µg/plate

- TA1537:   ± S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

- TA100 :    ± S9: 2500, 1250, 625, 313 ,156 and 78.1 µg/plate

Toxicity was observed at the highest or two highest dose levels with TA1537 tester strain both in the absence and presence of S9 metabolism and with TA100 only in its absence. 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 following dose levels were used:

- TA1535, WP2 uvrA: ± S9: 10000, 5000, 2500, 1250 and 625 µg/plate

- TA100: :+  S9: 10000, 5000, 2500, 1250 and 625 µg/plate

- TA1537, TA100: - S9: 5000, 2500, 1250, 625, 313  and 156 µg/plate

- TA1537:  +S9: 10000, 5000, 2500, 1250, 625  and 313 µg/plate

- TA98:  ± S9: 10000, 5000, 2500, 1250, 625 and 313 µg/plate

Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at the highest dose level tested with TA1537 and TA100 tester strains both in the absence and presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period in any experiment, at any concentration tested, 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 HH-2015-623 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.