Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
- Name of test material (as cited in study report): EXP1200078
- Physical state: dark brown viscous liquid
- Storage condition of test material: at room temperature in the dark

- Analytical purity: 100%
- Lot/batch No.: E00275-350
- Expiration date of the lot/batch: 31 Dec 2013

Method

Target gene:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100,
TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as
described by Green and Muriel (1976). Salmonella tester strains were derived from Dr. Bruce
Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine
Bacteria, Aberdeen, Scotland.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to
histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted
by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that
cause both frameshift and basepair substitution mutations. Specificity of the reversion
mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift
mutations (Green and Muriel, 1976).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Vehicle / solvent:
EXP1200078 - tetrahydrofuran (CAS 109-99-9)
Positive controls - dimethyl sulfoxide (CAS 67-68-5)
Water (Sodium Azide control)
Controls
Untreated negative controls:
yes
Remarks:
Water, dimethyl sulfoxide, ethanol, acetone
Negative solvent / vehicle controls:
yes
Remarks:
Water, dimethyl sulfoxide, ethanol, acetone
True negative controls:
no
Positive controls:
yes
Remarks:
Table of Positive Controls for Study AD63KG.503.BTL included below.
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene (CAS 613-13-8)
Details on test system and experimental conditions:
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock
into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested
in late log phase, the length of incubation was controlled and monitored. Following inoculation,
each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and
incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each
culture was monitored spectrophotometrically for turbidity and was harvested at a percent
transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual
titers were determined by viable count assays on nutrient agar plates.

Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was
prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™
1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot
No. 3017, Exp. Date: 17 October 2014) was purchased commercially from Moltox (Boone,
NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk
preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and
2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was
prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™
1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot
No. 3017, Exp. Date: 17 October 2014) was purchased commercially from Moltox (Boone,
NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk
preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and
2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
Evaluation criteria:
Initial Toxicity-Mutation Assay
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory
mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control,
positive controls and eight dose levels of the test article were plated, two plates per dose, with
overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal
agar in the presence and absence of Aroclor-induced rat liver S9.

Confirmatory Mutagenicity Assay
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential
of the test article. Five dose levels of test article along with appropriate vehicle control and
positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and
WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver
S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.
Statistics:
means and standard deviations

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, test article EXP1200078 was concluded to be negative in the
Bacterial Reverse Mutation Assay. The increase observed with tester strain TA1537 in the
presence of S9 activation in the initial toxicity-mutation assay was not considered indicative of
mutagenic activity because (1) the increase was non-dose responsive and not reproducible and
(2) all of the plate counts for each treated dose level were within the historical vehicle control
range. Therefore, the test article was concluded to be negative in this assay.