Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 July to 11 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA97, TA98, TA100, and TA1535), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA.
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Dose range finding test: A cytotoxicity screen was conducted in the S. typhimurium TA-100 strain at 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 µl/plate with and without S9-mix
Final test: 0.05, 0.1, 0.5, 1 and 5 µl/plate (with and without S9-mix).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tissue culture water (Sigma, Lot #RNBB7887) and DMSO (Fisher, Lot #12509, used as solvent for positive control 2AA)
- Justification for choice of solvent/vehicle: the test article was dissolved in tissue culture water and a clear solution was obtained.
Controls
Untreated negative controls:
yes
Remarks:
Tissue culture water (TCH2O)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: ICR-191 [1], Daunomycin hydrochloride [2] and 2-aminoanthracene [3]
Remarks:
with and without metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for the toxicity screen and the final mutation test.

DURATION
- Exposure duration: 48 - 72 hours
- Incubation: 37 ± 2°C

NUMBER OF REPLICATIONS: 3 plates/dose/strain. Positive and vehicle controls were run concurrently for all five strains, on six plates per strain.

NUMBER OF CELLS EVALUATED: all revertants per plates are counted using an Alphalmager 2200 fluorescence imager.

OTHER EXAMINATIONS:
- Other: Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytoxicity of the test article. The plates were also examined visually for test article precipitation.
Evaluation criteria:
A 2-fold increase of the mean number of revertant colonies 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, taking into account cytoxicity of the test article as well as the quality checks of the assay.
Statistics:
Statistical methods were not used in the determination of test results.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed on the plates in the cytotoxicity screen or the main assay.

RANGE-FINDING: yes
There was no diminuation and clearing of the background lawn observed at any dosage, indicating that the test article was not cytotoxic to baceterial strain TA-100 at 0.001 to 5 µl/plate.

QUALITY CHECKS:
- Sterility Test: Solutions and reagents used in the assay were tested for sterility in the main assay. Samples were added to minimal glucose agar plates and incubated at 37 ± 2°C for 48-72 hours. No contaminating organisms were detected in any of the reagents used in the assay.
- Vehicle controls: The spontaneous reversion rate, as presented by the mean colony forming units (CFU), for each strain of baceteria was measured and compared to in-house historical ranges. All vehicle controls passed the quality checks.
- Positive controls: The increase in revertants due to positive control treatment for each tester strain of bacteria was calculated (see Table 1 below). All positive controls passed the quality check.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Any other information on results incl. tables

Table 1: Summary Table of the Final Mutation Assay

Treatment

 

Mean values of revertants/Mutation factor (MF)

Salmonella thyphimurium tester strains

Escherichia coli

TA-97a

TA-98

TA-100

TA-1535

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Vehicle control

Mean

126.7

121.3

83.7

72.8

146.3

133.8

15.5

10.8

50.0

52.3

MF

-

-

-

-

-

-

-

-

-

-

5 µl/plate

Mean

119.0

130.7

78.0

83.7

150.7

165.7

14.0

13.0

47.0

48.3

MF

0.9

1.1

0.9

1.1

1.0

1.2

0.9

1.2

0.9

0.9

1 µl/plate

Mean

130.0

121.3

90.3

77.3

133.7

140.0

16.3

10.0

42.7

58.0

MF

1.0

1.0

1.1

1.1

0.9

1.0

1.1

0.9

0.9

1.1

0.5 µl/plate

Mean

127.0

116.7

87.3

73.7

142.3

145.3

10.7

12.7

53.3

57.3

MF

1.0

1.0

1.0

1.0

1.0

1.1

0.7

1.2

1.1

1.1

0.1 µl/plate

Mean

124.7

112.7

81.3

70.3

152.0

142.0

13.7

9.7

46.7

61.7

MF

1.0

0.9

1.0

1.0

1.0

1.1

0.9

0.9

0.9

1.2

0.05 µl/plate

Mean

116.3

125.3

76.7

78.7

135.3

121.0

13.0

10.3

58.3

54.0

MF

0.9

1.0

0.9

1.1

0.9

0.9

0.8

1.0

1.2

1.0

2AA (10 µg)

Mean

-

1421

-

1088.8

-

1011.0

-

185.3

-

201.0

MF

-

11.7*

-

15.0*

-

7.6*

-

17.2*

-

3.8*

ICR 191 (1 µg)

Mean

1474.7

-

-

-

-

-

-

-

-

-

MF

11.6*

-

-

-

-

-

-

-

-

-

NaN3 (1.5 µg)

Mean

-

-

-

-

465.0

-

312.2

-

-

-

MF

-

-

-

-

3.2*

-

20.1*

-

-

-

DM (6 µg)

Mean

-

-

600.5

-

-

-

-

-

-

-

MF

-

-

7.2*

-

-

-

-

-

-

-

MMS (2.5 µl)

Mean

-

-

-

-

-

-

-

-

390.7

-

MF

-

-

-

-

-

-

-

-

7.8*

-

*= 2-fold or more increase over vehicle control

Applicant's summary and conclusion

Conclusions:
Under the test conditions of this study, Tributyl(ethyl) phosphonium diethylphosphate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Tributyl(ethyl) phosphonium diethylphosphate was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to the OECD 471 Guideline and under GLP. The experiments

were carried out using four histidine-requiring strains of Salmonella typhimurium (TA 97, TA 98, TA 100 and TA 1535) and a tryptophan-requiring strain of Escherichia coli WP2uvrA in the presence and absence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix), using the plate incorporation method.

Based on the results of a solubility test, the test item was formulated in Tissue culture water. ln a cytotoxicity screen, the test item was tested at 0.001 to 5 µl/plate in the absence and presence of S9-mix in the strain TA100. The main assay was run in all five strains on triplicate plates at concentrations of 5, 1.0, 0.5, 0.1 and 0.05 μl/plate in the presence and absence of metabolic activation (S9). No reduced or clearing bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under the test conditions. There was no significant increase or dose-dependent increase of the number of revertants in any bacterial strain treated with the test article in the presence or absence of S9. All positive and negative controls values were within acceptable ranges, and all criteria for a valid study were met.

Based on the results of this study it is concluded that Tributyl(ethyl) phosphonium diethylphosphate does not have mutagenicity potential in the Bacterial Reverse Mutation Assay.