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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
2017-12-13 to 2018-02-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed on 2 November 2016
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
metabolic activation by a microsomal fraction of rat liver (S9-mix 10%v/v)
Test concentrations with justification for top dose:
- Concentrations for assay n°1: 50, 150, 500, 1500 and 5000 µg/plate
- Concentrations for assay n°2: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
Solutions were prepared in DMSO.
Controlsopen allclose all
Untreated negative controls:
yes
True negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Remarks:
Test item was completely soluble in ethanol at all concentrations tested.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Stock solution was formulated in DMSO.
Negative solvent / vehicle controls:
yes
Remarks:
NaCl 0.15 M
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Stock solution was formulated in NaCl 0.15 M.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Stock solution was formulated in DMSO.
Negative solvent / vehicle controls:
yes
Remarks:
NaCl 0.15 M.
Positive controls:
yes
Positive control substance:
other: cis-Platinium (II) Diammine Dichloride: for E. Coli without S9
Remarks:
Stock solution was formulated in NaCl 0.15M.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-anthramine: for TA1535, TA1537, TA98 and TA100 with S9
Remarks:
Stock solution was formulated in DMSO.
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Stock solution was formulated in acetone.
Details on test system and experimental conditions:
PREPARATION OF STOCK SOLUTION
-Test item is completely soluble in DMSO at all concentrations tested.
- Stock solution for each assay was prepared at 100 mg/mL in DMSO.
- Test item is tested at 5000, 1500, 500, 150 and 50 µg/plate.
- 50µL volume additions of test item solution were used for all treatments.

BACTERIA
- Strains of Salmonella typhimurium and Eschericia coli are purchased from Moltox. They are maintained in LEMI laboratory.
- The genotype of bacterial strains was checked.

EXPERIMENTS
- Triplicate plates test material.
- Negative, positive and vehicle controls were included in triplicate with and without metabolic activation.
- S9 fraction, microsome fraction, prepared from Sprague Dawley rat liver homogenate, was provided by Moltox USA.
- For experiments without metabolic activiation: 0.1 mL bacterial culture, 50µL of test article solution or control and 0.5 mL of buffer solution were added to 2 mL overlay agar at 45°C containing 10%v/v of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains and 5%v/v of nutrient broth n°2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/L for Escherichia coli strain. Plates are incubated at 37°C over 48-72 hour period.The pre-incubation phase is performed for the second assay if the first assay is negative.
- For experiments with metabolic activation: protocol similar to that described above, except that 500 µL of S9-mix fraction is quickly added before pouring the mixture onto the plates. Since the first assay was negative, the pre-incubation test was performed for the second assay.

COLONY COUNTING
- After a 48-72 hour incubation period at 37°C, revertant colonies are counted in each plate.
Evaluation criteria:
Ensure that the criteria of validity of the study are well respected namely:
- the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75%,
- the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory,
- negative and positive values should not show significant difference with the historical values of the laboratory (+/- 2 standard deviations).
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and E. coli WP2 (uvrA) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependant relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA98, TA100 and E. coli WP2 (uvrA) (pKM 101) and three fold for TA1535 and TA1537.
All results must be confirmed in an independant experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for positive controls (with and without metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (5000, 1500, 500, 150 and 50 µg/plate) without and with metabolic activaiton in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2(uvrA)(pKM101).
We can observe for all strains tested a significant decrease in the number of revertant colonies in the presence of highest dose tested 5000 µg/plate.
Results are confirmed in an independant experiment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
Doses (5000, 1500, 500, 150 and 50 µg/plate) performed from solutions of the test item do not induce any mutagenic change in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Eschirichia coli WP2(urA-)(pKM101) without, or with metabolic activation, according to the OECD guidelines n°471.