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Genetic toxicity: in vitro

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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:
03 April 2014 to 02 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & US EPA test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
other: liquid
Details on test material:
Identity: H2925 (CAS No. 68130-53-0), Lot# 2013090401Supplied by: Chemtura CorporationDate Received: 18 Mar 2014Storage: Room temperature and humidity.Description: Clear yellow liquid

Method

Target gene:
his operon (S. typhimurium strains)
trp operon ( E. coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA97a, TA1535, TA98 and TA100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
A cytotoxicity screen was conducted in the Salmonella typhimurium TA-100 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µI/plate) of the test article, two plates per dose.
Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5 µI/plate) of the test article were tested in each of five bacterial tester strains (E. coliWP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100).
Vehicle / solvent:
Identity: Acetone, Lot#124017
Supplied by: Fisher Scientific
Date Received: 15 Feb 2013
Expiration Date: Jul 2017
Storage: Room temperature.
Description: Clear colorless liquid
Sample Preparation: Used as received.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-Aminoanthracene (2AA), Acridine, 6-chloro-9-(3-((2-chloroethyl)amino)propyl)amino-2-methoxy, dihydrochloride (ICR-191), Daunomycin (DM),
Details on test system and experimental conditions:
Sample Preparation
Screen: 50 µI of the test article were brought to a volume of 1 ml with Acetone to yield a 50 µI/ml solution (stock for a dose of 5 µl/plate) (clear colorless liquid). Additional dilutions were made with Acetone (clear colorless liquids).
Main Assay: 500 µI of the test article were brought to a volume of 10 ml with Acetone to yield a 50 µI/ml solution (stock for a dose of 5 µl/plate) (clear colorless liquid). Additional dilutions were made with Acetone (clear colorless liquids).
Independent Repeat Assay: 500 µI of the test article were brought to a volume of 10 ml with Acetone to yield a 50 µl/ml solution (stock for a dose of 5 µI/plate) (clear colorless liquid). Additional dilutions were made with Acetone (clear colorless liquids).

Preparation of the Tester Strains: Bacterial cultures were inoculated by the addition of a lyophilized disk of each tester strain to Oxoid No.2 nutrient broth (Molecular Toxicology, Inc. (Moltox) Boone, NC, cat. #26-555). Ampicillin was added to the nutrient broth to ensure the retention of R-factor plasmid in tester strains TA-97a, TA-98 and TA-100. The cultures were incubated at 37°C ± 2°C with agitation. The cultures were used after they reached the late exponential growth phase as determined by absorbance readings at 600 nm.

Exogenous Metabolic Activation: An activation buffer containing 10% S9 obtained from the livers of Aroclor 1254-treated adult Sprague Dawley rats was prepared according to the manufacturer's (Moltox) instructions. Each vial of Regensys B (Moltox cat# 60-201) was reconstituted with approximately 1 ml of Regensys A (Moltox cat# 60-200). The solution was transferred back into the Regensys A bottle S9 (Moltox cat# 11-101) was then mixed with Regensys A+B to yield a 10% S9 buffer stock solution. The stock was separated into aliquots in sterile 15 ml conical tubes and refrigerated at 2-8°C until used.
The exogenous metabolic activation mixture was added to one set of all doses - each test article concentration, vehicle control and positive control for each of the bacterial tester strains.

Treatment of the Test System: Top agar supplemented with appropriate amino acids were prepared, as 2 ml aliquots, and maintained at 45-50°C in sterile culture tubes. Dulbecco's Phosphate Buffered Saline (DPBS) was added to the tubes not undergoing S9 activation (i.e. without S9, or -S9) to maintain equal dosing volumes. 0.1 ml of bacteria was added to the top agar, followed by 0.1 ml of the test article, vehicle control or positive control. For the activation portion of the test, 0.5 ml of S9 mixture was added last. The contents were gently vortexed and overiaid onto minimal glucose agar plates. After the mixture had solidified, the plates were incubated at 37°C ± 2°C for 48-72 hours. Plates that were not scored immediately following the incubation period were stored at 2-8°C until scoring.
An independent repeat (confirmatory) test was dosed using a preincubation method. One-hundred microliters of the test article concentration or Control was preincubated with 0.1 ml of the bacteria tester strain, along with 0.5 ml of Phosphate Buffered Saline or the metabolic activation (S9) system. These mixtures were incubated for 20 minutes or more at approximately 3rC prior to being mixed with the overlay agar and poured onto the surface of minimal agar plates. At the request of the Sponsor, the independent repeat (confirmatory) test was aborted after the plates were dosed.

Screen: Prior to the cytotoxicity screen, solubility of the test article was checked in tissue culture water (TCH2O), Dimethyl sulfoxide (DMSO), and Acetone. The test article was freely soluble at a concentration of 50 µI/ml only in Acetone and the Study Director chose Acetone as the vehicle for the study A cytotoxicity screen was conducted in the TA-100 tester strain using eight concentrations (0.001,0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µI/plate) of the test article, two plates per concentration, with and without S9. A vehicle control (Acetone) was run concurrently, with and without S9. The plates were incubated at 37°C ± 2°C for 48-72 Hours

Main Assay: Five concentrations (0.05, 0.1, 0.5, 1 and 5 µI/plate) of the test article were tested in each of five bacterial tester strains (Escherichia coli WP2 uvrA, and Salmonella typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Two sets of culture plates were dosed per concentration (+S9 and -S9). A vehicle control and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ± 2°C for 48-72 hours.

Revertant Colony Count: Counting of the revertants per plate was performed using an Alphalmager™ 2200 (Alpha Innotech Corporation, San Leandro, CA) fluorescence imager. Proper function of the imager was verified against a standard template (e.g. high (1000), medium (100) and low (10) counts) prior to each daily use. The number of revertants was recorded, along with observations of cytotoxicity. Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytotoxicity of the test article.
The plates were also examined visually for test article precipitate.

Independent Repeat Assay: The guidelines recommend that equivocal results be clarified by further testing, preferably using a modification of experimental conditions (e.g. concentrations tested), and that negative results need to be confirmed on a case-by-case basis. Justification should be provided when confirmation of negative results is not considered necessary. There is no recommendation of an independent repeat assay when the assay results are clearly positive.
At the request of the Sponsor, an independent repeat assay (confirmatory test) was aborted after all tester strains had been dosed with test material and plated but before the plates were scored.

Quality Check of the Assay:
Sterility Test: Sterile technique is required throughout the course of the study. To ascertain each component's sterility, a small amount (1 ml) was placed on agar plates and incubated to encourage growth of any contaminating microorganisms. The plates were incubated for 48-72 hours at 37°C ± 2°C and visually observed for microbial growth.
Vehicle Control: The spontaneous reversion rate, as represented by the mean colony forming units (CFU), for each strain of bacteria was calculated and compared to in-house historical ranges.
Positive Control: Positive control treatment for each tester strain of bacteria must result in at least a 2-fold increase of revertants over the mean vehicle control value. The effectiveness of the exogenous metabolic activation mixture was demonstrated by the positive response of the control.
Rationale for test conditions:
In accordance with test guidelines
Evaluation criteria:
Plates were scored based on the number of revertant colony-forming units present per plate. The number of revertants of each test article plate were averaged and plotted versus concentration of the test article. The mean number of revertants of each dose was divided by the mean for the vehicle control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test article as well as quality checks of the assay were taken into account.
In general, a 2-fold increase with or without metabolic activation is considered a positive response. Dose-related increases approaching a 2-fold increase are deemed equivocal.
A negative result is determined by the absence of a dose-related increase in all five tester strains, again taking into account cytotoxicity of the test article as well as the quality checks of the assay.
Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested strains.
Statistics:
Not specified in the study report.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA97a, TA1535, TA98 and TA100
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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:
not examined
Positive controls validity:
valid
Additional information on results:
Screen: There was no diminution or clearing of the background lawn observed at any dose level, indicating that the test article was not cytotoxic to tester strain TA-100 at 0.001 to 5 µI/plate. The Study Director chose 5 µI/plate as the top test article concentration for the main test.

Main Assay: The assay was run in all five strains on triplicate plates, Positive and vehicle controls were run concurrently for all five strains, on six plates per strain. All plating was with and without exogenous metabolic activation, S9. No reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under test conditions. There was no significant increase or dose-dependent increase of the number of revertants in any tester strain treated with the test article in the presence or absence of S9, All Positive and Negative Control values were within acceptable ranges, and all criteria for a valid study were met. However, TA-98 (with and without S9) and WP2 uvrA (with S9) initially did not pass QC parameters and had to be repeated. TA-98 with S9 did not pass the negative control QC parameters (average revertant colonies higher than historical range) but passed positive control parameter (greater than 2-fold increase over negative control), TA-98 without S9 did not pass either QC parameter and WP2 uvrA with S9 did not pass the positive control QC parameter, Upon repeating dosing for these two strains (TA-98 with and without S9, and WP2 uvrA with S9) all QC parameters were within acceptable ranges.

Independent Repeat Assay: An independent repeat assay was dosed in all five tester strains using test article concentrations of 0.05, 0.1, 0.5, 1, and 5 µI/plate and the pre-incubation dosing method. However, at the request of the Sponsor the assay was terminated before the plates were scored.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1

Test Article: H2925 (CAS No. 68130-53-0), Lot# 2013090401

Tester Strain: E.coli EP2 uvrA

Treatment

S9

CFU

Mean

Std. Dev.

Std. Error of Mean

Old Increase over Vehicle

Acetone (Vehicle Control)

+

63

50

51

48

43

52

51.3

6.5

2.7

 

5µl/plate

+

49

61

61

 

57.0

6.9

4.0

1.1

1µl/plate

+

47

65

54

55.32

9.1

5.2

1.1

0.5µl/plate

+

57

55

60

57.3

2.5

1.5

1.1

0.1µl/plate

+

47

54

61

54.0

7.0

4.0

1.1

0.05µl/plate

+

47

53

53

51.0

3.5

2.0

1.0

10µg 2AA (Positive Control)

+

132

131

166

152

121

134

141.0

14.7

6.0

2.7*

Acetone (Vehicle control)

-

73

41

98

98

119

83

85.3

26.7

10.9

 

5µl/plate

-

49

107

38

 

64.7

37.1

21.4

0.8

1µl/plate

-

95

94

75

88.0

11.3

6.5

1.0

0.5µl/plate

-

62

76

94

77.3

16.0

9.3

0.9

0.1µl/plate

-

45

59

68

57.3

11.6

6.7

0.7

0.05µl/plate

-

69

50

37

52.0

16.1

9.3

0.6

2.5µl MMS (Positive Control)

-

238

440

421

394

368

338

366.5

72.7

29.7

4.3*

*= 2-fold or more increase over Vehicle Control

 

Table 2

Test Article: H2925 (CAS No. 68130-53-0), Lot# 2013090401

Tester Strain: S. typh. TA-97a

Treatment

S9

CFU

Mean

Std. Dev.

Std. Error of Mean

Old Increase over Vehicle

Acetone (Vehicle Control)

+

63

54

68

61

81

88

69.2

12.9

5.3

 

5µl/plate

+

57

78

73

 

69.3

11.0

6.3

1.0

1µl/plate

+

75

80

80

78.3

2.9

1.7

1.1

0.5µl/plate

+

72

71

77

73.3

3.2

1.9

1.1

0.1µl/plate

+

77

75

59

70.3

9.9

5.7

1.0

0.05µl/plate

+

75

65

53

64.3

11.0

6.4

0.9

10µg 2AA (Positive Control)

+

611

564

541

497

496

468

529.5

52.8

21.5

7.7*

Acetone (Vehicle control)

-

40

52

46

58

63

56

52.5

8.4

3.4

 

5µl/plate

-

54

69

59

 

60.7

7.6

4.4

1.2

1µl/plate

-

57

59

65

60.3

4.2

2.4

1.1

0.5µl/plate

-

56

57

82

68.3

13.1

7.5

1.3

0.1µl/plate

-

69

58

65

64.0

5.6

3.2

1.2

0.05µl/plate

-

66

69

47

60.7

11.9

6.9

1.2

1µg ICR191 (Positive control)

-

1044

984

936

930

1091

1023

986.3

77.3

31.5

18.8*

*= 2-fold or more increase over Vehicle Control

 

Table 3

Test Article: H2925 (CAS No. 68130-53-0), Lot# 2013090401

Tester Strain: S. typh. TA-1535

Treatment

S9

CFU

Mean

Std. Dev.

Std. Error of Mean

Old Increase over Vehicle

Acetone (Vehicle Control)

+

10

8

10

13

12

9

10.3

1.9

0.8

 

5µl/plate

+

9

16

11

 

12.0

3.6

2.1

1.2

1µl/plate

+

5

10

9

8.0

2.6

1.5

0.8

0.5µl/plate

+

8

10

11

9.7

1.5

0.9

0.9

0.1µl/plate

+

10

11

14

11.7

2.1

1.2

1.1

0.05µl/plate

+

12

8

9

9.7

2.1

1.2

0.9

10µg 2AA (Positive Control)

+

47

44

36

44

48

52

45.2

5.4

2.2

4.4*

Acetone (Vehicle control)

-

11

12

7

7

16

8

10.2

3.5

1.4

 

5µl/plate

-

17

14

13

 

14.7

2.1

1.2

1.4

1µl/plate

-

8

13

6

9.7

2.9

1.7

1.0

0.5µl/plate

-

9

14

12

11.7

2.5

1.5

1.1

0.1µl/plate

-

11

5

12

9.3

3.6

2.2

0.9

0.05µl/plate

-

11

5

10

8.7

3.2

1.9

0.9

1.5µg NaN3(Positive Control)

-

545

548

534

552

515

482

529.3

26.7

10.9

51.9*

*= 2-fold or more increase over Vehicle Control

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under test conditions, test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, did not have mutagenicity potential.
Executive summary:

Objective: The purpose of this study is to evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation. This protocol is based on OECD Guideline for Testing of Chemicals: No. 471 - Bacterial Reverse Mutation Test and U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 - Bacterial Reverse Mutation Test.

 

Method Synopsis: Prior to the cytotoxicity screen, solubility of the test article was checked in tissue culture water (TCH2O), Dimethyl sulfoxide (DMSO), and Acetone. The test article was freely soluble at a concentration of 50µI/mlonly in Acetone and the Study Director chose Acetone as the vehicle for the study. A cytotoxicity screen was conducted in the Salmonella typhimurium TA-100 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5µI/plate)of the test article, two plates per dose. The test article was combined with the bacteria and top agar in the presence and absence of a metabolic activation mixture (S9) and overlaid onto minimal glucose agar plates. An Acetone vehicle control was run concurrently, with and without S9.

Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5µI/plate)of the test article were tested in each of five bacterial tester strains(E.coliWP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Vehicle controls and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ± 2°C for 48-72 hours. Revertant colony growth was determined by counting the colonies per plate using an Alphalmager™ imaging system. The number of revertants of the test article treatment plates and positive control plates was divided by the number of revertants of the vehicle plates. In general, a positive result is determined by a 2-fold increase above the vehicle control.

Tester strains WP2 uvrA (with S9) and TA-98 (with and without S9) failed the quality checks. The main test was repeated for these strains and passed the quality checks. The results of the repeat main test are reported; the original data is maintained in the study file.

At the request of the Sponsor, an independent repeat assay (confirmatory test) was aborted after all tester strains had been dosed with test material and plated but before the plates were scored.

 

Summary: Test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, in the vehicle, Acetone, was tested in a Bacterial Reverse Mutation Assay. In the screen, the test article did not show obvious cytotoxicity to tester strain TA-100 at any concentration, with or without S9. In the main test, the test article at 0.05 to 5µI/plate,with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, indicating that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay.

 

Conclusion: Under test conditions, test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, did not have mutagenicity potential.