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
Study period:
2008-02-15 to 2008-03-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Minor deviation from guideline: - justification for choice of vehicle was not clearly provided. It was only stated that the vehicle was selected based on information given by the sponsor.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008-05-31
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-10-15
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Molecular formula: C14H25NO3
- Molecular weight: 255.36g/mol
- Physical state: clear colourless extremely viscous liquid
- Storage conditions: room temperature in the dark

Method

Target gene:
not applicable
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from the livers of male Sprague-Dawley rats, which had received phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1:
- all strains without S9: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- all strains with S9: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Additional dose levels (5 and 15 µg/plate were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

Experiment 2:
- all strains without S9: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
- all strains with S9: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
An additional dose level (15 µg/plate) was again included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test material was fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. Dimethylsulphoxide was therefore selected as the vehicle.

The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment.
Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 X 10°-4 microns.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation; Strains TA100 and TA1535

Migrated to IUCLID6: 3 µg/plate for TA100 and 5 µg/plate for TA1535
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic acitivation; strain TA1537

Migrated to IUCLID6: 80 µg/plate
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation; strain TA102

Migrated to IUCLID6: 0.5 µg/plate
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation; strain TA98

Migrated to IUCLID6: 0.2 µg/plate
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2- Aminoanthracene; 1 µg/plate for TA100 and 2 µg/plate for TA1535 and TA1537
Remarks:
with metabolic activation, strain TA100
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation; strain TA98

Migrated to IUCLID6: 5 µg/plate
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-dihydroxyanthraquinone; 10 µg/plate
Remarks:
with metabolic activation; strain TA102
Details on test system and conditions:
Experiment 1 and 2:
METHOD OF APPLICATION: in agar (plate incorporation)
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.

DURATION
- Exposure duration: all of the plates were incubated at 37°C for approximately 48 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: the frequency of revertant colonies were assessed using a Domino colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
A preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.The test was performed by mixing 0.1 mL of bacterial culture (TA100), 2 mL of molten, trace histidine supplemented, top agar, 0.1 mL of test material formulation, 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). ten doses of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

ACCEPTABILITY CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are present in the "Attached background material" below with historical control ranges.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 X 10^9 bacteria per mL.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges are presented in "Attached background material" below.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation.
Cytotoxicity:
yes
Remarks:
The test material was toxic at and above 1500 and at 5000 µg/plate, in the absence and presence of S9, respectively
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test material was toxic to the strain of Salmonella used (TA100) at and above 1500 and at 5000 µg/plate, in the absence and presence of S9, respectively. The test material formulation and the S9 -mix used in this experiment were both shown to be sterile. The number of revertant colonies for the toxicity can be seen in table 1 in the field "Any other information on results incl. tables" below.

COMPARISON WITH HISTORICAL CONTROL DATA: a history profile of vehicle and positive control value was given in the study report.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused a visible reduction in the growth of the bacterial background lawns and/or substantial decrease in the frequency of revertant colonies to all of the tester strains in the presence and absence of S9 at 5000 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

NEGATIVE CONTROL.
Results for the negative controls (spontaneous mutation rates) are presented in in table 2 in the field "Any other information on results incl. tables" below. They were acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation test.

Any other information on results incl. tables

Table 1

With (+) or without (-) metabolic activation

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

78

75

80

70

93

88

90

92

84

73S

30SP

+

TA100

96

88

82

87

90

76

89

81

87

98

36SP

SP: Sparse bacterial background lawn

P: Precipitate

Table 2 Spontaneous Mutation Rates (concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

86

88            (87)

86

16

19                 (18)

20

241

266             (260)

273

25

15                 (19)

17

15

15                 (15)

16

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

102

146            (124)

123

29

26                 (32)

40

375

309             (352)

371

16

25                (19)

15

15

13                 (14)

15

RESULTS POSITIVE CONTROLS:

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.