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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:
27 February 2018 - 15 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
n/a
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
n/a
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
The selected dose levels were as follows:
- 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1537 (first experiment) and the TA 1535, TA 98 and TA 100 strains (both experiments),
- 0.34, 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1537 (second experiment),
- 31.25, 62.5, 125, 250, 500 and 1000 µg/plate in the TA 102 strain (both experiments).

Experiments with S9 mix
The selected dose levels were as follows:
- 250, 500, 1000, 2000 and 5000 µg/plate in the TA 1535, TA 98, TA 100 and TA 102 strains (first experiment using the direct incorporation method),
- 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate in the TA 1537 (first experiment using the direct incorporation method),
- 2.3, 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate in the TA 1537 (second experiment using the pre-incubation method)
- 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1535, TA 98 and TA 100 strains (second experiment using the pre-incubation method),
- 2.06, 6.17, 18.5, 55.6, 167 and 500 µg/plate in the TA 102 strain (second experiment using the pre-incubation method).
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); The preliminary test, all experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: observation of a decrease in number of revertant colonies and/or a thinning of the bacterial lawn.
Rationale for test conditions:
Not applicable.
Evaluation criteria:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
. a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
. and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
. neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
. nor any evidence of a dose-response relationship is noted.
Statistics:
no

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
other: No genotoxicity was obtained with the TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Emulsion

RANGE-FINDING STUDY:
A moderate emulsion was noted at dose levels = 500 µg/plate without S9 mix and at dose levels = 2500 µg/plate with S9 mix in the TA 98, TA 100 and TA 102 strains.
A moderate to strong toxicity was noted at dose levels = 100 µg/plate without S9 mix and at 5000 µg/plate with S9 mix, in both the TA 98 and TA 100 strains. No noteworthy toxicity was noted in the TA 102 strain, either with or without S9 mix.

RESULTS OF CYTOTOXICITY and GENOTOXICITY:
A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels = 500 µg/plate in the TA 102 strain without S9 mix and at dose levels = 2000 µg/plate in the TA 1535, TA 98, TA 100 and TA 102 strains with S9 mix (direct incorporation method).

Experiments without S9 mix:
In the first experiment, a moderate to strong toxicity was noted at dose levels = 27.8 µg/plate in the TA 1535 and TA 1537 strains and at dose levels = 83.3 µg/plate in the TA 98 and TA 100 strains.
In the second experiment, a slight to strong toxicity was noted at dose levels = 27.8 µg/plate in the TA 1535, TA 98 and TA 100 strains and at 250 µg/plate in the TA 1537 strain.
No noteworthy toxicity was noted towards the TA 102 strain, in both experiments.

Experiments with S9 mix:
In the first experiment (using the direct plate incorporation method), a slight to moderate toxicity was noted at 5000 µg/plate in the TA 1535 and TA 98 strains, at dose levels = 555.6 µg/plate in the TA 1537 strain and = 2000 µg/plate in the TA 100 strain. No noteworthy toxicity was noted towards the TA 102 strain in the first experiment.
In the second experiment (using the pre-incubation method), a slight to strong toxicity was noted at 250 µg/plate in the TA 1535 and TA 100 strains, at dose levels = 185.2 µg/plate in the TA 1537 strain, = 83.3 µg/plate in the TA 98 strain and = 500 µg/plate in the TA 102 strain.


The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, either with or without S9 mix. These results met thus the criteria of a negative response.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains (i.e. TA 1535, TA 1537, TA 98, TA 100 and TA 102), either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

 

The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose levels of the test item, dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The treatments of the TA 1537 strain [first experiment with S9 mix and second experiment (with and without S9 mix)] were performed at the test site.

Results

The test item was freely soluble in the vehicle at 100 mg/mL.

Consequently, using a maximum dose-volume of 50 µL/plate, the dose-levels used for the preliminary toxicity test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

 

Preliminary test results:

A moderate emulsion was noted at dose levels = 500 µg/plate without S9 mix and at dose levels = 2500 µg/plate with S9 mix in TA 98, TA 100 and TA 102 strains.

 In both the TA 98 and TA 100 strains, a moderate to strong toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at dose levels >= 100 µg/plate, without S9 mix and at 5000 µg/plate (decrease in the number of revertants), with S9 mix. No noteworthy toxicity was noted in the TA 102 strain, either with or without S9 mix.

Since the test item was found toxic and poorly soluble in the final treatment medium in the preliminary test, the selection of the highest dose level to be used in the main experiments was based on the level of toxicity and/or presence of emulsion, according to the criteria specified in the international guidelines.

 

Main experiments:

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

  

Experiments without S9 mix

The selected dose levels were:

.  1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1535, TA 98 and TA 100 strains for both mutagenicity experiments and in the TA 1537 strain for the first experiment,

. 0.34, 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1537 strain for the second experiment,

. 31.25, 62.5, 125, 250, 500 and 1000 µg/plate in the TA 102 strain for both mutagenicity experiments.

 

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose levels = 500 µg/plate in the TA 102 strain in both experiments.

In the first experiment, a moderate to strong toxicity was noted at dose levels = 27.8 µg/plate in the TA 1535 and TA 1537 strains and at dose levels = 83.3 µg/plate in the TA 98 and TA 100 strains.

In the second experiment, a slight to strong toxicity was noted at dose levels = 27.8 µg/plate in the TA 1535, TA 98 and TA 100 strains and at 250 µg/plate in the TA 1537 strain.

No noteworthy toxicity was noted towards the TA 102 strain, in both experiments.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Experiments with S9 mix

The selected dose levels were:

. 250, 500, 1000, 2000 and 5000 µg/plate for the first mutagenicity experiment in the TA 1535, TA 98, TA 100 and TA 102 strains,

. 6.9, 20.6, 61.7, 185.2, 55.6, 1666.7 and 5000 µg/plate in the TA 1537 for the first experiment,

. 2.3, 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate in the TA 1537 for the second experiment,

. 2.06, 6.17, 18.5, 55.6, 167 and 500 µg/plate in the TA 102 strain for the second mutagenicity experiment,

. 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1535, TA 98 and TA 100 strains for the second experiment.

 

In the first experiment, a moderate emulsion was observed in the Petri plates when scoring the revertants at dose levels = 2000 µg/plate in the TA 1535, TA 98, TA 100 and TA 102 strains.

In the first experiment (using the direct plate incorporation method), a slight to moderate toxicity was noted at 5000 µg/plate in the TA 1535 and TA98 strains, at dose levels = 555.6 µg/plate in the TA 1537 strain and = 2000 µg/plate in the TA 100 strain. No noteworthy toxicity was noted towards the TA 102 strain in the first experiment.

In the second experiment (using the pre-incubation method), a slight to strong toxicity was noted at 250 µg/plate in the TA 1535 and TA 100 strains, at dose levels = 185.2 µg/plate in the TA 1537 strain, = 83.3 µg/plate in the TA 98 strain and = 500 µg/plate in the TA 102 strain.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, either with or without S9 mix. These results met thus the criteria of a negative response.

 

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.