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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 November 2008 to 25 December 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Remarks:
"GLP Standards for the Test Facility on New Chemical Substances" (Notification No. 1121003 of Pharmaceutical and Food Salfety Bureau, Ministry of Health, Labour and Welfare, No.3 of Manufacturing Industries Bureau. Ministry of Economy, Trade and Industry,
Type of assay:
bacterial reverse mutation assay

Test material

Method

Target gene:
Histidine operon was used for the S. typhimurium TA strains (Salmonella typhimurium TAl 00, Salmonella typhimurium TA 1535 (base pair substitution), Salmonella typhimurium TA98 and Salmonella typhimurium TA 1537 (frame shift)).

Tryptophan operon was used for the Escheria coli WP2 uvrA strain (base pair substitution).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate

Main test: 5000 µg/plate
Vehicle / solvent:
- solvent used: water

- Justification for choice of solvent: Based on the information from the sponsor that the test substance was soluble in water at 20% and more.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix

Migrated to IUCLID6: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of Escherichia coli.
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate.
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix

Migrated to IUCLID6: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix

Migrated to IUCLID6: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-fury)acrylamide: 0.01 µg/plate,
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9 mix

Migrated to IUCLID6: 0.5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-fury)acrylamide: 0.01 µg/plate
Remarks:
Without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-fury)acrylamide: 0.1 µg/plate
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine∙2HCl: 1.0 µg/plate.
Remarks:
without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: The culture tube was left at 4°C for 7 hours and 45 minutes.

SELECTION AGENT (mutation assays): Not applicable.
Evaluation criteria:
In the dose-finding test and the main test, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed. The test substance was to be judged positive. Statistical analysis was not done.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the dose-finding test and the main test, neither an increase in the number of revenant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3SD) in background data, indicating that this study was performed correctly.

From these results, mutagenicity of the test substance was judged negative.

The growth inhibition of the test strains by the test substance was not observed. and the precipitate on the plates was not observed either with or without metabolic activation.

In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

References

1) B.N. Ames, F.D. Lee, and W.E. Durston: An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens, Proc. Natl. Acad. Sci. USA Vo1.70, No.3, pp.782-786, March 1973

2) J. McCann, N. E. Spingarn, J. Kobori and B.N. Ames: Detection of Carcinogens as Mutagens: Bacterial Tester Strains with R Factor Plasmids, Proc. Natl. Acad. Sci. USA Vol.72, No.3, pp.979-983, March 1975

3) M.H.L. Green and W.J. Muriel: Mutagen Testing using Trp+ Reversion in Escherichia coli, Mutation Res. Vol.38, pp.3-32. 1976

4) T. Yahagi, M. Nagao, Y. Seino. T.Matsushima. T.Sugimura and M.Okada: Mutagenicities of N-nitrosamines on Salmonella, Mutation Res. Vol.38, pp.121-130, 1977

5) D.Maron and B.N. Ames: Revised Methods for the Salmonella Mutagenicity Test, Mutalion Res. Vol.l13, pp.173-215.1983

6) A guidebook for Mutagenicity Studies using Bacteria, New Edition, Ed. by Chemical Substances Investigation Division, Industrial Safety and Health Department, Ministry of Labor, Japan Industrial Safety and Health Association, 1986

7) Testing Methods for Environmental Mutagens: Ed. by Tajima, Kada, Kondo and Tamura, Koudan-sha Co., pp.56-68. 1980

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

From the results described above, it is concluded that H-MI is not mutagenic in the bacterial reverse mutation
assay carried out under the experimental conditions.
Executive summary:

Mutagenicity potential of H-Ml was assessed with Salmonella typhimurium TA 100, TA 1535, TA98, TA 1537 and Escherichia coli WP2 uvrA. In this study, neither an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor a dose-related response was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activalion.

From the above, it is judged H-MI has no mutagenicity to bacteria under the described study conditions.