Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-27 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on June 17, 2015 / Signed on September 24, 2015)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
Brown red solid
Details on test material:
- Storage condition of test material: Room temperature, in the dark

Method

Target gene:
Histidine and tryptophan for S. typhimurium and E. coli, respectively.
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):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
Test concentrations with justification for top dose:
Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method:
- E.coli strain WP2uvrA and Salmonella strain TA98 (absence and presence of S9-mix) and Salmonella strain TA100 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
- Salmonella strains TA1535 and TA1537 (absence and presence of S9-mix) and Salmonella strain TA100 (absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test formulation: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. No correction was made for purity. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM
- Bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. Manual counts were performed at 5000 μg/plate because of a test item film. A number of further manual counts were also required due to colonies spreading and artefacts on the plates, thus distorting the actual plate count. Occasional plates were also manually assessed for accuracy against the automated counts.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and 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 uvr A
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2013 and 2014 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of TA100 at 5000 μg/plate in the both the presence and absence of S9-mix. Small reductions in revertant colony frequency were also noted for TA98 at 5000 μg/plate (absence of S9-mix) and for TA1537 at 5000 μg/plate (absence and presence of S9-mix). No further toxicity was noted to any of the remaining bacterial strains. Consequently, for the second mutation test the maximum recommended dose level of 5000 μg/plate was again employed as the maximum dose concentration for all of the bacterial tester strains. Results from the second mutation test (pre-incubation method) exhibited weakened bacterial background lawns in the absence of S9-mix to TA1537 from 500 μg/plate, to TA100 from 1500 μg/plate and to TA98 at 5000 μg/plate. In the presence of S9-mix, weakened bacterial background lawns were noted at 5000 μg/plate to TA1537 only, although slight reductions in WP2uvrA revertant colony frequency were noted at the same maximum dose concentration. No further significant toxicity was noted to any of the remaining bacterial strains dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. An opaque test item film was noted by eye at 5000 μg/plate; this observation did not prevent the scoring of revertant colonies.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar, S9-mix and test item formulation used in both experiments were shown to be sterile.

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA-.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method:

- E.coli strain WP2uvrA and Salmonella strain TA98 (absence and presence of S9-mix) and Salmonella strain TA100 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

- Salmonella strains TA1535 and TA1537 (absence and presence of S9-mix) and Salmonella strain TA100 (absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

 

Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

 

In Experiment 1, the test item induced a visible reduction in the growth of the bacterial background lawns of TA100 at 5000 μg/plate in the both the presence and absence of S9-mix. Small reductions in revertant colony frequency were also noted for TA98 at 5000 μg/plate (absence of S9-mix) and for TA1537 at 5000 μg/plate (absence and presence of S9-mix). No further toxicity was noted to any of the remaining bacterial strains. In Experiment 2, weakened bacterial background lawns in the absence of S9- mix to TA1537 from 500 μg/plate, to TA100 from 1500 μg/plate and to TA98 at 5000 μg/plate. In the presence of S9-mix, weakened bacterial background lawns were noted at 5000 μg/plate to TA1537 only, although slight reductions in WP2uvrA revertant colony frequency were noted at the same maximum dose concentration. No further significant toxicity was noted to any of the remaining bacterial strains dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. An opaque test item film was noted by eye at 5000 μg/plate; this observation did not prevent the scoring of revertant colonies.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in any of the experiments.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA-.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.