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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-10-2013 to 29-10-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Reaction mass of 1-hydroxydecan-3-one and 3-(hydroxymethyl)nonan-2-one and nonan-2-one
EC Number:
909-017-6
Molecular formula:
C10H20O2 C9H28O
IUPAC Name:
Reaction mass of 1-hydroxydecan-3-one and 3-(hydroxymethyl)nonan-2-one and nonan-2-one
Test material form:
liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
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. 3130, Exp. Date: 01 August 2015) 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. The S9 mix was prepared on the day of use: β-nicotinamide-adenine dinucleotide phosphate (4 mM), Glucose-6-phosphate (5 mM), Potassium chloride (33 mM), Magnesium chloride (8 mM), Phosphate Buffer (pH 7.4) (100 mM), S9 homogenate (10% (v/v)). The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was also prepared on the day of use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.
Test concentrations with justification for top dose:
Initial Toxicity-Mutation Assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate
Confirmatory Mutagenicity Assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (-S9: TA100, TA1535 and WP2 uvrA; +S9: TA98 and TA1535)
Confirmatory Mutagenicity Assay: 15, 50, 150, 500, 1500 and 5000 μg/plate (remaining test conditions)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was conducted to determine the vehicle. The test was conducted using water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other:
Details on test system and experimental conditions:
TEST SYSTEM
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 0.3E09 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

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 substance 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 substance. Six or seven dose levels of test substance 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 substance, vehicle control and positive controls were plated in triplicate.

PLATING AND SCORING PROCEDURES
The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). On the day of its use, minimal top agar, containing 0.8% agar (W/V) and 0.5% NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM 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. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5% (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5% (W/V) agar and supplemented with 2.5% (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5% (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Each plate was labeled with a code system that identified the test substance, test phase, dose level, tester strain and activation, as described in detail in BioReliance's Standard Operating Procedures. One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted. The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown below:
- 1 or no code: Normal: Distinguished by a healthy microcolony lawn.
- 2: Slightly Reduced: Distinguished by a noticeable thinning of the microcolony lawn and possibly a slight increase in the size of the microcolonies compared to the vehicle control plate.
- 3: Moderately Reduced: Distinguished by a marked thinning of the microcolony lawn resulting in a pronounced increase in the size of the microcolonies compared to the vehicle control plate.
- 4: Extremely Reduced: Distinguished by an extreme thinning of the microcolony lawn resulting in an increase in the size of the microcolonies compared to the vehicle control plate such that the microcolony lawn is visible to the unaided eye as isolated colonies.
- 5: Absent: Distinguished by a complete lack of any microcolony lawn over greater than or equal to 90% of the plate.
- 6: Obscured by Particulate: The background bacterial lawn cannot be accurately evaluated due to microscopic test substance particulate.
- NP: Non-Interfering Precipitate: Distinguished by precipitate on the plate that is visible to the naked eye but any precipitate particles detected by the automated colony counter total less than or equal to 10% of the revertant colony count (e.g., less than or equal to 3 particles on a plate with 30 revertants).
- IP: Interfering Precipitate: Distinguished by precipitate on the plate that is visible to the naked eye and any precipitate particles detected by the automated colony counter exceed 10% of the revertant colony count (e.g., greater than 3 particles on a plate with 30 revertants). These plates are counted manually.
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.
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.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and 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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Initial Toxicity-Mutation Assay: No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Confirmatory Mutagenicity Assay (See tables under "Any other information on results"): No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg per plate.

Any other information on results incl. tables

Confirmatory Mutagenicity Assay without S9 activation: Mean revertants per plate

 

TA98

TA100

TA1535

TA1537

WP2uvrA

DMSO

14

85

9

9

19

5 µg/plate

 -

91

10

-

22

15 µg/plate

15

92

11

7

20

50 µg/plate

10

93

11

8

13

150 µg/plate

11

93

8

10

19

500 µg/plate

17

86

8

7

24

1500 µg/plate

8

55

6

5

20

5000 µg/plate

0

0

0

0

0

Confirmatory Mutagenicity Assay with S9 activation: Mean revertants per plate

 

TA98

TA100

TA1535

TA1537

WP2uvrA

DMSO

18

116

15

9

22

5 µg/plate

25

-

14

-

-

15 µg/plate

25

102

21

8

20

50 µg/plate

24

94

10

11

22

150 µg/plate

24

98

10

10

26

500 µg/plate

20

95

11

10

26

1500 µg/plate

17

78

7

6

18

5000 µg/plate

0

0

0

0

0

Applicant's summary and conclusion

Conclusions:
The test material was considered non-mutagenic under the conditions of this test.
Executive summary:

The test substance was tested in the Bacterial Reverse Mutation Assay, performed according to OECD TG 471 and following GLP, 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 using the plate incorporation method. DMSO was selected as the solvent based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test. In the initial toxicity-mutation assay, the dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate with tester strains TA100, TA1535 and WP2 uvrA in the absence of S9 activation and tester strains TA98 and TA1535 in the presence of S9 activation and 15, 50, 150, 500, 1500 and 5000 μg per plate with the remaining test conditions. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg per plate. Under the conditions of this study, the test substance was concluded to be negative in the Bacterial Reverse Mutation Assay.