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Genetic toxicity: in vitro

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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:
7th April 2011 to 22nd May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP compliant study performed in line with appropriate guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
- Physical state: off-white solid
- Storage condition of test material: room temperature in the dark

Method

Target gene:
Histidine operon in Salmonella species
Tryptophan operon in Escherichia species
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient broth
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient broth
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate produced from the liver of rats induced with Phenobarbitone/ß-Naphthoflavone orally at 80/100 mg/kg/day
Test concentrations with justification for top dose:
Experiments 1 and 2 - 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: insoluble in distilled water, acetone, dimethyl formamide and acetonitrile at 50 mg/mL. Forms a partial solution in DMSO.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: and 9-aminoacridine (9AA), 4-nitroquinoline-1-oxide (4NQO), 2-aminoanthracene (2AA) and benzo(a)pyrene (BP)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

MUTATION TEST - EXPERIMENT 1
0.1 mL of the appropriate bacterial culture was dispensed into a test tube along with 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test substance, vehicle or positive control and either 0.5 mL S9-mix or phosphate buffer. The contents of each tube were mixed and distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). The procedure was repeated in triplicate for each bacterial strain for each test substance concentration with and without S9-mix. All plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 µg/plate because of excessive test substance precipitation.

MUTATION TEST - EXPERIMENT 1
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by either 0.5 mL S9-mix or phosphate buffer and 0.1 mL of the vehicle or test substance and incubated for 20 minutes at 37 ºC with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of each tube were mixed and distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). The procedure was repeated in triplicate for each bacterial strain for each test substance concentration with and without S9-mix. The positive and untreated controls were dosed as detailed for Experiment 1. All plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 µg/plate because of excessive test substance precipitation.

ACCEPTANCE CRITERIA
The assay is considered valid if the following criteria are met:
- all bacterial strains demonstrate the required characteristics as determined by their respective strain checks;
- all tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls;
- all tester strain cultures should be in the range of 0.9 - 9.0 x 10E+9 bacteria per mL;
- positive controls should induce marked increases in the frequency of revertant colonies with and without metabolic activation;
- there should be a minimum of four non-toxic dose levels; and
- there should be no evidence of excessive contamination.
Evaluation criteria:
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;
- a reproducible increase at one or more concentrations;
- biological relevance against in-house historical control ranges;
- statistical analysis of data; or
- greater than two-fold increase for any tester strain against concurrent solvent controls.

A test substance will be considered non-mutagenic if the above criteria are not met.
Statistics:
Statistical analysis of data was determined by UKEMS.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A small increase in revertant colony frequency was observed in TA1535 (in the presence of S9 mix) at 50 µg/plate in Experiment 1 and TA100 (in the absence of S9 mix) at 1500 µg/plate in Experiment 2. Neither of these increases were greater than twice the concurrent solvent control value and they were not reproducible in a separate experiment. There was also no evidence of a dose response relationship and the observed counts were within the historical range. These increases were therefore not considered to be of biological or toxicological significance.

See figures 3 and 4 below.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A pale precipitate was noted at and above 1500 µg/plate in experiment 1 and 500 µg/plate in experiment 2. This did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: Prior to use the master strains were checked for characteristics, viability and spontaneous reversion rate. All were found to be satisfactory. The top agar and S9-mix were found both to be sterile. The culture density for each strain was considered to be acceptable. Results from the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the mutation test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number of revertant colonies in preliminary test

 with (+) or without (-) S9 mix Strain Dose (µg/plate)
    0 0.15 0.5 1.5 5 15 50 150 500 1500 5000
 - TA100 102 88 91 113 97 102 93 92 89 105P 106P
 + TA100 82 97 100 94 93 104 93 89 85 84P 96P
 - WP2uvrA 31 37 38 38 27 33 34 26 32 33P 29P
 + WP2uvrA 32 27 32 33 37 39 27 32 34 34P 40P

P = precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the study the test substance was considered to be non-mutagenic.
Executive summary:

In a GLP compliant bacterial reverse mutation study conducted in line with OECD Guideline 471, EU Method B.13/B.14 and EPA OPPTS 870.5265, the genotoxicity of the test substance was determined using the 'Ames' test. The substance was considered to be non-mutagenic.

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