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Toxicological information

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:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): QM-1326AP
- Lot/batch No.: 2506828
- Storage condition of test material: Stored in the dark at room temperature

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:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 pg per plate
Vehicle / solvent:
dimethyl sulfoxide (DMSO, CAS No. 67-68-5)
Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the
test article and compatibility with the target cells.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: The test system was exposed to the test article via the plate incorporation methodology.

On the day of its use, minimal top agar, containing 0.8 % agar (WN) and 0.5 % NaCl (WN),
was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final
concentration of 50 pM each. Top agar not used with S9 or Sham mix was supplemented with
25 mL of water for each 100 mL of minimal top agar. For the preparation of media and
reagents, all references to water imply sterile, deionized water produced by the Milli-Q Reagent
Water System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956)
containing 1.5 % (WN) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E
containing 1.5 % (WN) agar and supplemented with 2.5 % (WN) Oxoid Nutrient Broth No. 2
(dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (WN)
Oxoid Nutrient Broth No. 2 (dry powder).

Overnight cultures were prepared by inoculating 'from the appropriate master plate or from the
appropriate frozen permanent stock into a vessel containing -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 resting shakerlincubator at room
temperature. The shakerlincubator 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 approximately lo9 cells per milliliter. The actual titers were determined by
viable count assays on nutrient agar plates.

One-half (0.5) milliliter of S9 or Sham mix, 100 yL of tester strain and 50 yL of vehicle or test
article dilution were added to 2.0 rnL of molten selective top agar at 45*2OC. After vortexing,
the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the
positive controls, the test article aliquot was replaced by a 50 yL aliquot of appropriate positive
control. After the overlay had solidified, the plates were inverted and incubated for
approximately 48 to 72 hours at 37*2OC. Plates that were not counted immediately following
the incubation period were stored at 2-8OC until colony counting could be conducted.
The condition of the bacterial background lawn was evaluated for evidence of test article
toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination
without magnification. Toxicity and degree of precipitation were scored relative to the vehicle
control plate.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: approximately 10E9 cells per milliliter

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, revertant colonies
Evaluation criteria:
For the test article 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 article.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: sparingly soluble in water, but soluble in DMSO

RANGE-FINDING/SCREENING STUDIES: 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 uwA on selective minimal
agar in the presence and absence of Aroclor-induced rat liver S9.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control articles have been characterized as per the Certificates of
Analysis on file with the testing facility. The stability of the negative and positive control
articles and their mixtures was demonstrated by acceptable results that met the criteria for a
valid 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: Average Revertants Per Plate ± Standard Deviation

Liver Microsomes: None

 Dose (µg/plate)  TA98  TA100  TA1535  TA1537  WP2 uvrA
 Vehicle  18 ± 2  94 ± 23  33 ± 12  12 ± 3  16 ± 4
 50  24 ± 10  103 ± 32  35 ± 4  11 ± 1  17 ± 4
 150  23 ± 1  78 ± 10  42 ± 3  12 ± 2  17 ± 3
 500  21 ± 4 106 ±  31  29 ± 4  15 ± 4  9 ± 4
 1500  17 ± 0  84 ± 10  33 ± 9  13 ± 7  11 ± 4
 5000  20 ± 5  92 ± 11  23 ± 2  9 ± 4  19 ± 0
 Positive  134 ± 29  638 ± 188  248 ± 40  362 ± 144  71 ± 4

Liver Microsomes: Rat liver S9

 Dose (µg/plate) TA98   TA100  TA1535  TA1537  WP2 uvrA
 Vehicle  29 ± 8  105 ± 12  22 ± 3  11 ± 2  15 ± 1
 50  34 ± 10  115 ± 17  20 ± 5  17 ± 5 19 ±
 150  28 ± 8  127 ± 22  21 ± 3  14 ± 5  16 ± 5
 500  33 ± 1  135 ± 6  23 ± 6  13 ± 3  18 ± 3
 1500  19 ± 4  107 ± 9  22 ± 5  10 ± 3  14 ± 5
 5000  28 ± 2  100 ± 26  19 ± 3  12 ± 2  22 ± 4
Positive  180 ± 8  381 ± 31  70 ± 11  46 ± 9  128 ± 61

Vehicle = Vehicle Control              

Positive = Positive Control (50 µL plating aliquot)

Plating aliquot: 50 µL

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results of the Bacterial Reverse Mutation Assay (Ames Assay) indicate that, under the conditions of this study, QM-1326AP did not cause a positive response in either the presence or absence of Aroclor-induced rat liver S9.
Executive summary:

The test article, QM-1326AP, was tested in the Bacterial Reverse Mutation Assay (Ames Assay) using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a soluble and clear solution in dimethyl sulfoxide (DMSO) at approximately 500 mg/mL, the highest concentration tested.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 pg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. In the initial toxicity-mutation assay, no positive mutagenic response was observed. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the initial toxicity mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 pg per plate.

In the confirmatory mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 50, 150, 500, 1500 and 5000 pg per plate. Neither precipitate nor appreciable toxicity was observed. Under the conditions of this study, test article QM-1326AP was concluded to be negative in the Bacterial Reverse Mutation Assay (Ames Assay).