Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 3rd February 2017 to 20th February 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)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

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:
Standard S9 mix (rat) and Reductive S9 mix-Prival modification (Syrian hamster)
Test concentrations with justification for top dose:
-Toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate; the choice was based on results of the performed solubility test.
-Main Assay I and II: 5000, 2500, 1250, 625, 313 (for all strains in assays I and II) and 156 µg/plate (only for TA1537 strain in assay I); the choice was based on the results obtained in the preliminary toxicity test.
Vehicle / solvent:
Sterile water for injection. Water was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity.
Controls
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
methylmethanesulfonate
other: 2-Aminoanthracene in DMSO and Trypan blue Solution 0.4%
Details on test system and experimental conditions:
PRELIMINARY TOXICITY TEST
Solubility of the test item was evaluated in a preliminary trial using water for injection. In the preliminary test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatment was performed both in the absence and presence of S9 metabolism (Rat Mixed Induction) 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 ASSAYS
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.

-Main Assay I
The test was performed using a plate-incorporation method and the standard metabolic activation system. 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. The overlay mixture was composed by: 2.0 ml of overlay agar (held at 45°C), 0.1 ml of test or control item solution, 0.5 ml of S9 mix or phosphate buffer (pH 7.4, 0.1 M), 0.1 ml of bacterial suspension.
-Main Assay II
Based on the chemical structure of the test item (azo-dyes), the test was performed using a pre-incubation method and the reductive metabolic activation system (Prival modifi cation). The components were added in turn to an empty
test-tube were: 0.1 mL of bacterial suspension, 0.1 ml of test or control item solution, 0.5 ml of modified S9 mix or phosphate buffer (pH 7.4, 0.1 M) and, when it was necessary, 0.05 ml of 2-Aminoanthracene solution. The incubate was vortexed and placed at 37 °C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

INCUBATION AND SCORING
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, plates from the Main Assays were held at 4 °C for 24 hours, while plates from the toxicity test were immediately evaluated. Plates were scored by counting the number of revertant colonies and analysing the background lawn on each plate.

BACTERIAL STRAINS
Permanent stocks of these strains are kept at -80°C. 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.
Bacterial cultures in liquid and on agar were clearly identified with their identity.

MEDIA
-Nutrient Broth: 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: 25 g of Nutrient Broth No and 15 g of Difco Bacto-agar were added to 1 l of distilled water and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes, allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
-Minimal Agar: was prepared as 1.5 % Difco Bacto-agar in Vogel-Bonner Medium E, with 2 % Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
- Top Agar: was prepared as 0.6 % Difco Bacto-agar + 0.5 % NaCl in distilled water and autoclaved. Prior to use, 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 ml).

S9 TISSUE HOMOGENATE
The two different S9 tissue fractions were provided by Trinova Biochem GmbH and the producer was MOLTOX, Molecular Toxicology.
-Standard (rat): liver tissue of rat (Sprague Dawley) and Phenobarbital – 5,6-Benzoflavone as liver inducing agent were used. The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared for each 10 ml with S9 tissue fraction (1.0 ml), NADP (0.4 ml, 100 mM), G-6-P (0.5 ml, 100 mM), KCl (1.0 ml 330 mM), MgCl2 (0.8 ml, 100 mM), Phosphate buffer (5.0 ml, pH 7.4, 200 mM) and Distilled Water (1.3 ml).
-Reductive (Prival modification): liver tissue of Syrian hamster (GSH) and none inducing agent were used. The modified mixture of S9 was prepared for each 10 ml with Phosphate buffer (4.85 ml, pH 7.4, 0.2 M), KCl (0.25 ml, 1.32 M), MgCl2.6H2O (0.25 ml, 0.32 M), FMN (0.5 mol, 40 mM), NADH (0.25 ml, 80 mM), NADP (0.4 ml, 100 mM), G-6-P dehydrogenase (0.25 ml, 112 U/mL), G-6-P (0.25 ml, 800 mM) and Uninduced sirian hamster S9 tissue fraction (3.0 ml).
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:
The regression analysis fited 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
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
Regression lines were calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines were also given.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA1535, TA1537, TA98, TA100 and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA1535, TA1537, TA98, TA100 and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity, as indicated by thinning of the background lawn, was observed at the highest dose level in the absence of S9 Mix with TA1537 and TA100 tester strains.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
SOLUBILITY
The test item was found to be soluble at 50.0 mg/ml.

TOXICITY TEST
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. Plates treated with the test item presented a dose dependent brown colour of the agar, which did not interfere with the scoring of colonies. A slight reduction in revertant numbers, probably indicative of a toxic effect, was observed with TA1537 tester strain at the highest dose level, in the absence of S9 metabolism. No relevant increases in revertant colonies were observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

MAIN ASSAYS
-Main Assay I: Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.
-Main Assay II: No relevant increase in revertant numbers was observed at any dose level, with any tester strain, in the absence or presence of S9 metabolic activation.

In both assays, plates treated with the test item presented a dose dependent brown colour of the agar (brown-yellow in the presence of the Prival modified metabolic system), which did not interfere with the scoring of colonies.
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.

EVALUATION
Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.
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 did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system.
Remarks on result:
other: Main Assay I

Applicant's summary and conclusion

Conclusions:
The test item did 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 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) and Escherichia coli (WP2 uvrA), as measured by reversion of auxotrophic strains to prototrophy. The test item was used as a solution in sterile water for injection. 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 (standard metabolic activation) in Main Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification), in Main Assay II.

The test item was assayed in the toxicity test at concentrations of 5000, 1580, 500, 158 and 50.0 µg/plate.

Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at dose levels of 5000, 2500, 1250, 625, 313 (for all strains) and 156 µg/plate (only for TA1537).

At the end of the incubation period, no relevant toxicity of the test item was seen, with any tester strain, at any dose level, in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed. Based on the chemical structure of the test item (azo-dyes), the experiment was performed using the pre-incubation method in the presence of a reductive metabolic system (hamster S9 supplemented with flavin mononucleotide cofactor). The test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate. Slight toxicity was observed in the absence of S9 metabolic activation at the highest dose level with TA1537 and TA100 tester strains.

No precipitation of the test item 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, in any experiment.

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 any metabolic activation system.

Based on results of these experiments, the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coliin the absence or presence of S9 metabolism.