Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
From the 21st to the 27th of May, 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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Separate formulations of the test item were prepared, immediately before use, in sterile water for injection at 41.4 and 13.1 (25.0 and 7.91 mg/mL in terms of active ingredient) for the preliminary toxicity test, and at 41.4 and 20.7 mg/mL (25.0 and 12.5 mg/mL in terms of active ingredient) for the Main Assay. Vortexing and/or sonication at 37°C for approximately 30 minutes were employed to achieve a satisfactory formulation. Suspensions/solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item. All test item solutions were used within 1 hour and 25 minutes from the initial formulation. All dose levels in this report are expressed to three significant figures.

Method

Target gene:
Reverse mutation assays employ bacterial strains which are already mutant at a locus whose phenotypic effects are easily detected.
The Salmonella tester strains have mutations causing dependence on a particular amino acid (histidine) for growth.
The ability of test items to cause reverse mutations (reversions) to histidine-independence can easily be measured.
The E. coli tester strains of the WP2 series are similarly mutant at the tryptophan locus.


Since many chemicals only demonstrate mutagenic activity after metabolism to reactive forms, in order to detect these "indirect mutagens" the test is performed in the presence and absence of a rat liver metabolising system. In addition, for azo-dyes and diazo-compounds, a reductive metabolic activation system prepared with S9 liver fraction from uninduced hamsters, supplemented with flavin mononucleotide cofactor is used (Prival et al. 1982 and 1984).
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):
TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens.
In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds.
The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface
of the bacterial cell wall and increases permeability to certain classes of chemicals.
All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens.
TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.
Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens.
In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.
Metabolic activation:
with and without
Metabolic activation system:
S9 and Privall
Test concentrations with justification for top dose:
2500, 1250, 625, 313, 156 µg/plate, with and without standard metabolic activation system.
Vehicle / solvent:
Sterile water
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
sterile water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and water
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
Absence S9
Untreated negative controls:
yes
Remarks:
sterile water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and water
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Presence of S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
congo red
other: 2-aminoanhtracene, Trypan Blue
Remarks:
Presenceof S9, Reductive metabolic system
Details on test system and experimental conditions:
Permanent stocks of these strains are kept at -80°C in the laboratory.
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 on Minimal plates+Biotin.
Growth on Minimal plates+Biotin+Histidine.
Tryptophan requirement:
No Growth on Minimal agar plates.
Growth on Minimal 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.

MEDIA
Nutrient Broth
Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
Nutrient Agar
Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
Minimal Agar
Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-BonnerMedium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
Top Agar
"Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5mM tryptophan) was added to the top agar (100 mL).

S9 tissue homogenate
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics: Rat, Strain Sprague Dawley, Liver, Inducing Agents Phenobarbital – 5,6-Benzoflavone, Producer MOLTOX,Molecular Toxicology, Inc., Batch Number 3647
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared to 10.0 mL.

S9 mix Privall modification (Main Assay II) Species Syrian hamster, Strain GSH, Tissue Liver, Inducing Agents None, Producer MOLTOX,Molecular Toxicology, Inc., Batch Number 3342. The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared to 10.0 mL.

PRELIMINARY TOXICITY TEST
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assay. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included.
Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.
MAIN ASSAY
Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The experiment was performed using a plate-incorporation method and the standard metabolic activation system (Rat-Mixed Induction). The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar
and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.

Incubation and scoring
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were immediately scored by counting the number of revertant colonies and analysing the background lawn on each plate.
Evaluation criteria:
Acceptance criteria
The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays
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.

Evaluation
The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.
The test item induced two-fold and more increases in the number of revertant colonies, both in the absence and presence of S9 metabolism. A statistically significant dose-related relationship was indicated. Since a clear positive response was observed, no further experiment was undertaken.
Statistics:
The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as: y = a +bx

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Dose related increases in the number of revertant colonies (all doses).
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Neither toxicity, nor precipitation. Dose dependent brown colour of the agar, which did not interfere with the scoring of colonies. Large and dose-related increases in revertant colonies in absence and presence of S9 metabolism.
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:
ambiguous
Remarks:
Dose related increases in the number of revertant colonies (all doses).
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Neither toxicity, nor precipitation. Dose dependent brown colour of the agar, which did not interfere with the scoring of colonies. Large and dose-related increases in revertant colonies in absence and presence of S9 metabolism.
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:
positive
Remarks:
Dose related increases in the number of revertant colonies (all doses).
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Neither toxicity, nor precipitation. Dose dependent brown colour of the agar, which did not interfere with the scoring of colonies. Large and dose-related increases in revertant colonies in absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Dose related increases in the number of revertant colonies (only at the highest dose).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Neither toxicity, nor precipitation. Dose dependent brown colour of the agar, which did not interfere with the scoring of colonies. Large and dose-related increases in revertant colonies in absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility
Solubility of the test item was evaluated in a preliminary trial using distilled water and DMSO. These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. An opaque formulation with slight precipitation, suitable for treatment but not for serial dilutions, was obtained in water for injection at 41.4 mg/mL (25.0 mg/mL in terms of active ingredient), following vortexing and sonication at 37°C for
approximately 30 minutes. An opaque formulation without any precipitation was obtained at 20.7 mg/mL (12.5 mg/mL in terms of active ingredient) and was considered suitable for serial dilutions.

Toxicity test
The test item was assayed in the toxicity test at a maximum dose level of 2500 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 791, 250, 79.1 and 25.0 µg/plate.
Neither toxicity, nor 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.
Plates treated with the test item presented a dose dependent brown colour of the agar, which did not interfere with the scoring of colonies.
Large and dose-related increases in revertant colonies were observed with almost all tester strains, both in the absence and presence of S9 metabolism.

Main assays
AMain Assay was performed. Individual plate counts for this test and the mean and standard error of the mean for each test point, together with a statistical analysis.
On the basis of the results obtained in the preliminary toxicity test, in theMain Assay, using the plate incorporation method, the test item was assayed at the following dose levels: 2500, 1250, 625, 313 and 156 µg/plate.
Neither toxicity, nor precipitation of the test item was observed at the end of the incubation period with any tester strain, at any dose level, in the absence or presence of S9 metabolic activation. Plates treated with the test item presented a dose dependent brown colour of the agar, which did not interfere with the scoring of colonies and evaluation of the background lawn.
Large and dose-related increases in revertant numbers were observed both in the absence and presence of S9 metabolism with all tester strains with the exception of TA1535, that showed a mild but biologically relevant increase only at the highest dose level in the presence of S9 metabolic activation.
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. 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.
Remarks on result:
other: Main Assay

Applicant's summary and conclusion

Conclusions:
The test item induced reverse mutation in Salmonella typhimurium tester strains both in the absence and presence of S9 metabolism, under the
reported experimental conditions.
Executive summary:

Method

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. 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/suspension in sterile water for injection. As indicated by the Sponsor, concentrations were expressed in terms of active ingredient.

Toxicity test

2500, 791, 250, 79.1 and 25.0 µg/plate, with and without standard metabolic activation system.

Main Assay I

2500, 1250, 625, 313, 156 µg/plate, with and without standard metabolic activation system.

Results

Toxicity test

At the end of the incubation period, neither toxicity, nor precipitation of the test item was observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Large and dose-related increases in revertant numbers were observed with almost all tester strains both in the absence and presence of S9 metabolism.

Main Assay I

Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.

Test item treatment yielded large and dose-related increases in revertant numbers, both in the absence and presence of S9 metabolic activation, with all tester strains with the exception of TA1535 that showed a mild but significant increase only in the presence of S9 metabolism. Since a clear positive response was observed, no further experiment was undertaken.

Conclusion

It is concluded that the test item induces reverse mutation in Salmonella typhimurium, both in the absence and presence of S9 metabolism, under the reported experimental conditions.