Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 31.Mar. 2017 to 24.Apr.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)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
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:
S9
Test concentrations with justification for top dose:
In Main Assay I and II, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625, and 313 µg/plate.
On the basis of the results obtained in the preliminary toxicity test, 5000 µg/plate was selected as the top concentration for the Main Assays in order to evaluate a potential mutagenic effect up to cytotoxic or insoluble concentrations.
As limited by solubility, the test item was assayed in the toxicity test at a maximum concentration of 2500 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 791, 250, 79.1 and 25.0 µg/plate.
Vehicle / solvent:
- Vehicles used:
Sterile water for injection (Baxter, batches 15I0211, 15I0302, 15G2401 and 15G2402).
Dimethylsulfoxide (DMSO) (Fluka AG, batch STBF8595V).
Controlsopen allclose all
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
Absence of S9
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Presence of S9, Standard metabolic activation system
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
congo red
other: Trypan blue and 2-aminoanthracene
Remarks:
Presence of S9, Reductive metabolic activation system
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Main Assay I was performed using a plate-incorporation method and the standard metabolic activation system (Rat-Mixed Induction).
Main Assay II was performed using a pre-incubation method and the reductive metabolic activation system (Prival modification).

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-Bonner Medium 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, 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)

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main 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 (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.

- OTHER:
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.
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
at an intermediate dose level (without S9), dose-related increases of 1.9-fold (with S9, Prival)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Main assay II at the the two highest dose levels without S9
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
at higher concentrations (without S9); dose-related increases 2.0-fold (with S9 Prival)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Main assay II at the the two highest dose levels without S9
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
a dose-related increase of 2.3-fold (prival)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY TEST
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.
Slight increases in revertant numbers were observed with TA100 tester strain in the absence of S9 metabolism and with TA1537 at the highest dose level in its presence. Plates treated with the test item presented a dose dependent green color of the agar, which did not interfere with the scoring.
SOLUBILITY
An opaque formulation without any precipitation was obtained in water for injection at 25.0 mg/ml following sonication at 37 °C for approximately 30 minutes and was considered suitable for serial dilutions.
PRECIPITATION
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.
STERILITY
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.
OTHER
In both assays, plates treated with the test item presented a dose dependent green color of the agar, which did not interfere with the scoring.

Any other information on results incl. tables

In 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. In the absence of S9 metabolism, an increase in revertant numbers (2.4-fold) was seen with TA1537 tester strain at an intermediate dose level. A slight increase was also observed with TA100 tester strain at higher concentrations. Based on these equivocal results, Main Assay II was performed using the Prival modification method and the same concentration range.

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. Toxicity, as indicated by thinning of the background lawn and reduction in revertant colonies, was observed with TA1537 and TA100 tester strains at the two highest dose levels in the absence of S9 metabolism.

Dose-related increases in revertant numbers were observed with TA1537 (1.9-fold), TA98 (2.3-fold) and TA100 (2.0-fold) tester strains in the presence of S9 metabolism. At higher dose levels, the number of revertant colonies fell out the current historical control range for negative controls. No relevant increases were observed with the remaining tester strains.

VALIDITY CRITERIA FULFILLED

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.

Applicant's summary and conclusion

Conclusions:
The test item induces reverse mutation in bacteria in the presence of a reductive metabolic activation system, 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 and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The test was performed according to the OECD Guideline 471 (1997) and the EU method B.13/14 of EC 440/2008. 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 (standard metabolic activation) in Main Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system using the Prival modification method), in Main Assay II. The test item was used as a solution/suspension in sterile water for injection. As limited by solubility, the test item was assayed in the toxicity test at a maximum concentration of 2500 µg/plate and at four lower concentrations.

On the basis of toxicity test results, in order to evaluate a potential mutagenic effect up to cytotoxic or insoluble concentrations, in the Main Assays an additional dose level of 5000 µg/plate was employed. In Main Assay I, using the plate incorporation method and the standard S9 metabolic activation system, the test item was assayed at five dose levels from 313 to 5000 µg/plate. 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.

As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was carried out using the pre-incubation method in the presence of a reductive metabolic system. The test item was assayed at the same dose levels. 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. Toxicity was observed with TA1537 and TA100 tester strains at the two highest dose levels in the absence of S9 metabolism. Dose-related and biologically relevant increases in revertant numbers were observed with TA1537, TA98 and TA100 tester strains in the presence of S9 metabolism. These increases were greater than twice the control values with TA98 and TA100 tester strains and so can be considered as clear evidence of mutation induction.

Thus, the test item induces reverse mutation in bacteria in the presence of a reductive metabolic activation system, under the reported experimental conditions.