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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to O.E.C.D. Testing Guideline No. 471 with GLP compliance.

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
As per IUCLID5 Sections 1.1. - 1.4.

Method

Target gene:
Histidine operon.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: and E. coli WP2 uvrA.
Metabolic activation:
with and without
Metabolic activation system:
Induced rat liver derived S9 fraction with cofactors.
Test concentrations with justification for top dose:
The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate with tester strain E. coli WP2 uvrA, 5.0, 15, 50, 150, 500 and 1500 μg per plate with Salmonella tester strains TA98 and TA1537 and 15, 50, 150, 500, 600, 750 and 1500 μg per plate with tester strains TA100 and TA1535.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
for with and without S9 metabolic activation.
Positive control substance:
other: 2-aminoanthracene, CAS No. 613-13-8, with S9 metabolic activation.
Remarks:
Tester strain specific positive controls without S9 metabolic activation were: 2-nitrofluorene, sodium azide, 9-aminoacridine and methyl methanesulfonate.
Details on test system and experimental conditions:
The Salmonella tester strains were received directly from Dr. Bruce Ames, University of California, Berkeley and the E. coli tester strain was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. Overnight cultures in approximately 50 mL of culture medium were initiated by placement in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37±2°C 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.

Aroclor 1254-induced rat liver S9 fraction was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 lot was prepared by and purchased from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 fraction was stored at -60°C or colder until used. The S9 fraction cofactors mix was prepared immediately before its use and contained 10% S9 fraction, 5 mM glucose-6-phosphate, 4 mM ß-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4.

Treatment of the bacterial tester strains was by the preincubation method. A minimum of five dose levels of Crude 1,3-dichloropropene along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA
on selective agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test substance, vehicle control and positive controls were plated in triplicate.

Minimal top agar for plating, contained 0.8 % agar (W/V) and 0.5 % NaCl (W/V) and was and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar.

For preincubation treatment one-half (0.5) milliliter of S9 fraction cofactors preparation or 100 mm phosphate buffer pH 7.4, 100 μL of tester strain and 50 μL of vehicle or test substance dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2°C. After vortexing, these mixtures were incubated with shaking for 60±2 minutes at 37±2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and
the mixture was vortexed and overlaid onto the surface of minimal bottom agar plates. After the overlay had solidified, the plates were inverted and
incubated for approximately 48 to 72 hours at 37±2°C. Revertant mutant colonies were counted with an automated colony counter.
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains TA1535 and TA1537 were judged positive if the
increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
Statistics:
No data

Results and discussion

Test results
Species / strain:
other: Strains TA 1535, TA 100 and WP2 uvrA.
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitate was observed up to the high dose level of Crude 1,3-dichloropropene of 5000 ug/plate. Toxicity was observed at dose levels of 1500 or at 5000 μg per plate. Positive mutagenic responses were observed with tester strains TA100, TA1535 and E. coli WP2 uvrA in the presence and absence of S9 metabolic activation preparation. The increase in the mutant frequency of E. coli WP2 uvrA was 2.2-2.7 -fold at 1500 ug/plate. The increase of the mutant frequency for tester strain TA 1535 was 16.2-fold without S9 metabolic activation and 31.4-fold with S9 metabolic activation preparation at 750 ug/plate. In tester strain TA 100 the maximum increase in the mutant frequency was 4.3 to 4.4-fold with and without S9 metabolic activation preperation.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

Treatment of tester strains TA 1535, TA 100 and WP2 uvrA with Crude 1,3-dichloropropene with and without rat liver derived S9 metabolic activation perperation resulted in positive mutational responses. Tester strain TA 1535 was the most sensitive indicator of the mutagenic effect of Crude 1,3-dichloropropene. The data suggest that Crude 1,3-dichloropropene is acting by the induction of point mutations in these bacterial tester strains.
Executive summary:

Crude 1,3 -dichloropropene was assessed for mutagenic potential with an O.E.C.D. 471 Testing Guideline study conducted by the preincubation method and GLP compliance. Treatment of tester strains TA 1535, TA 100 and WP2 uvrA with Crude 1,3-dichloropropene with and without rat liver derived S9 metabolic activation perperation resulted in positive mutational responses. Tester strain TA 1535 was the most sensitive indicator of the mutagenic effect of Crude 1,3-dichloropropene. An increase in the mutant frequency that reached 31.4 -fold the control background value was observed for strain TA 1535 with S9 metabolic activation preperation. The data suggest that Crude 1,3-dichloropropene is acting by the induction of point mutations in these bacterial tester strains.