Registration Dossier

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:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial gene mutation assay

Test material

Reference
Name:
Unnamed
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:
S9 fraction from uninduced hamsters supplemented with flavin mononucleotide cofactor (reductive metabolic activation system) was used in Main Assay II.
Test concentrations with justification for top dose:
Solubility of the test item was evaluated in a preliminary trial using sterile water for injection. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50.0mg/mL. This result permitted a maximum concentration of 5000 μg/plate to be used in the toxicity test

The test item was assayed in the toxicity test at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately half-log
intervals: 1580, 500, 158 and 50.0 μg/plate.
No precipitation of the test item was observed at the end of the incubation period at any concentration. 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.
Plates treated with the test item presented a dose dependent orange colour of the agar which did not interfere with the scoring
Vehicle / solvent:
Sterile water for injection
Dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
methylmethanesulfonate
other: 2-aminoanthracene, Trypan Blue
Details on test system and experimental conditions:
Bacterial strains
Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study.
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, as follows:
Histidine requirement No Growth on Minimal plates+Biotin.
Growth onMinimal plates+Biotin+Histidine.
Tryptophan requirement No Growth onMinimal agar plates.
Growth onMinimal 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
The following growth media were used:
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 0.5mMBiotin + 0.5mM Histidine (or 0.5mMtryptophan) was added to the top agar (100 mL).

S9 tissue homogenate
Two different S9 tissue fractions, provided by Trinova Biochem GmbH, were used in this study and had the following characteristics:
Species Rat
Strain Sprague Dawley
Tissue Liver
Inducing Agents Phenobarbital – 5,6-Benzoflavone
Producer MOLTOX,Molecular Toxicology, Inc.
Batch Number 4009

Species Syrian hamster
Strain GSH
Tissue Liver
Inducing Agents None
Producer MOLTOX,Molecular Toxicology, Inc.
Batch Number 3855


The standard mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows
(for each 10 mL):
S9 tissue fraction 1.0mL
NADP (100 mM) 0.4mL
G-6-P (100 mM) 0.5mL
KCl (330 mM) 1.0mL
MgCl2 (100 mM) 0.8mL
Phosphate buffer (pH 7.4, 200 mM) 5.0mL
DistilledWater 1.3mL
10.0mL

The modified mixture of S9 (S9 mix-Prival modification) used inMain Assay II was prepared as follows (for each 10 mL):
Phosphate buffer (pH 7.4, 0.2 M) 4.85 mL
KCl (1.32 M) 0.25 mL
MgCl2.6H2O (0.32 M) 0.25 mL
FMN (40 mM) 0.5 mL
NADH (80 mM) 0.25 mL
NADP (100 mM) 0.4 mL
G-6-P dehydrogenase (112 U/mL) 0.25 mL
G-6-P (800 mM) 0.25 mL
Uninduced syrian hamster S9 tissue fraction 3.0 mL
10.0mL


Preparation of the test item
Solutions of the test item, as received, were prepared immediately before use in sterile water for injection. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 1 hour and 48 minutes from the initial preparation.

Analysis of test item preparation
No assay of test item stability, nor its concentration and homogeneity in solvent were undertaken.

Preliminary toxicity test
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in theMain Assays. In this 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 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
Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plate 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 first Main Assay 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.

The overlay mixture was composed as follows:
Overlay agar (held at 45°C) 2.0mL
Test or control item solution 0.1mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
Bacterial suspension 0.1mL

The second Main Assay was performed using a pre-incubation method and the reductive metabolic activation system (Prival modification). The components were added in turn to
an empty test-tube:
Bacterial suspension 0.1mL
Test item solution 0.1mL
(Positive control item solution)* (0.05 mL)
Modified S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
* For Congo Red and Trypan Blue, 0.1 mL was added instead of 0.05 mL

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 were immediately scored by counting the number of revertant colonies on each plate
Rationale for test conditions:
Two Main Assays were performed. Individual plate counts for these tests and the mean and standard error of the mean for each test point, together with a statistical analysis.
The maximum concentration of the test item to be used in the main experiments should be determined taking into consideration cytotoxicity and solubility in the final treatment mixture. On the basis of the results obtained in the preliminary toxicity test, 5000 μg/plate (the upper limit to testing indicated in the Study Protocol) was selected as the maximum dose level for theMain Assays.
In Main Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625, and 313 μg/plate with all tester strains.
No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.
As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed using the pre-incubation method and the same concentrations.
Based on the chemical structure of the test item (azo-dye), in the presence of S9 metabolism a reductive metabolic system (uninduced hamster S9 supplemented with flavin mononucleotide cofactor) was used as described by Prival et al., 1984.
Evaluation 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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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 S9 metabolism.

Any other information on results incl. tables

Solubility

Solubility of the test item was evaluated in a preliminary trial using sterile water for injection.

This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50.0mg/mL. This result permitted a maximum concentration of 5000 μg/plate to be used in the toxicity test.

Toxicity test

The test item was assayed in the toxicity test at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate.

No precipitation of the test item was observed at the end of the incubation period at any concentration. 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.

Plates treated with the test item presented a dose dependent orange colour of the agar which did not interfere with the scoring.

Main Assays

Two Main Assays were performed. Individual plate counts for these tests and the mean and standard error of the mean for each test point, together with a statistical analysis.

The maximum concentration of the test item to be used in the main experiments should be determined taking into consideration cytotoxicity and solubility in the final treatment mixture. On the basis of the results obtained in the preliminary toxicity test, 5000 μg/plate (the upper limit to testing indicated in the Study Protocol) was selected as the maximum dose level for theMain Assays.

In Main Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625, and 313 μg/plate with all tester strains.

No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed using the pre-incubation method and the same concentrations.

Based on the chemical structure of the test item (azo-dye), in the presence of S9 metabolism a reductive metabolic system (uninduced hamster S9 supplemented with flavin mononucleotide cofactor) was used as described by Prival et al., 1984.

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.

No precipitation of the test item, evident to the unaided eye, was observed at the end of the incubation period at any concentration, in any experiment, in the absence or presence of S9 metabolism.

In both assays, plates treated with the test item presented a dose dependent orange colour of the agar (yellowin the presence of the reductive 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

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.
Executive summary:

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. A cofactor supplemented liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone (standard metabolic activation system) was used in the preliminary toxicity test and in Main Assay I, while, based on the chemical structure of the test item (azo-dyes), a liver S9 fraction from uninduced hamsters supplemented with flavin mononucleotide cofactor (reductive metabolic activation system) was used inMain Assay II.

The test item was used as a solution in sterile water for injection.

The test item was assayed in the toxicity test at a maximum concentration of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate.

No precipitation of the test item was observed at the end of the incubation period at any concentration. 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 the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625 and 313 μg/plate with all tester strains.

No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was carried out. 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) and the same concentrations of Main Assay I. Neither toxicity, nor relevant increase in the number of revertant colonies was observed at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period, at any concentration, 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.

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.