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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:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected August 2005; signature: November 2005
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydro-4-methyl-2-phenyl-2H-pyran
EC Number:
303-662-1
EC Name:
Tetrahydro-4-methyl-2-phenyl-2H-pyran
Cas Number:
94201-73-7
Molecular formula:
C12H16O
IUPAC Name:
4-methyl-2-phenyltetrahydro-2H-pyran
Test material form:
other: liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: In the dark at approximately 4°C under nitrogen.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.
Main test:
- Experiment 1 (range-finding test): Salmonella strains - 1.5, 5, 15, 50, 150, 500 and 1500 ug/plate; E.coli strain - 15, 50, 150, 500, 1500 and 500 ug/plate.
- Experiment 2 (main test): TA100 (without S9-mix): 5 to 1500 ug/plate; TA100 (with S9-mix), TA1535, TA98, TA1537: 15 to 15000 ug/plate; WP2uvrA·: 50 to 5000 ug/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): incubated at 37 degrees C for approximately 48 hours

NUMBER OF REPLICATIONS: 3
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. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity- this will be reported as equivocal.

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:
cytotoxicity
Remarks:
observed at 5000ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at 500ug/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No test material precipitate was observed .on the plates at any of the doses tested in either the presence or absence of S9-mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Range finding study

Range-finding test with and without S9
  Number of revertants (mean number of colonies per plate)
S9 Mix Test substance concentration (ug/plate) TA100 Mean TA1535 Mean WP2uvrA- Mean TA98 Mean TA1537 Mean
- 0 98 92 10 14 18 24 21 19 14 12
73 20 21 19 10
104 12 34 18 12
- 1.5 106 102 7 11 NT NT 29 17 13 14
109 10   13 15
92 15   10 13
- 5 86 94 12 10 NT NT 16 17 12 13
88 8   18 14
109 9   16 13
- 15 81 80 21 15 26 30 18 19 14 13
80 13 21 25 13
78 10 43 13 13
- 50 76 79 13 16 20 20 19 18 15 13
75 12 18 12 13
86 23 22 22 11
- 150 87 86 14 19 30 25 21 18 13 12
81 21 21 19 11
89 22 23 15 13
- 500 90* 92 21 21 24 20 31 22 9 10
95* 18 18 25 9
91* 25 19 11 12
- 1500 0* 0 0* 0 22 22 0* 0 0* 0
0* 0* 22 0* 0*
0* 0* 22 0* 0*
  5000 N/T N/T 19* 14 N/T N/T
13*
9*
                       
+ 0 77 75 8 10 32 33 18 23 22 23
74 14 37 20 24
74 7 30 30 24
+ 1.5 64 70 7 8 NT 26 27 14 19
78 10 41 24
67 8 14 20
+ 5 71 71 16 11 NT 25 23 21 19
77 8 24 12
66 8 19 23
+ 15 81 78 13 9 37 32 26 25 15 19
75 4 23 24 18
79 10 35 25 23
+ 50 79 72 7 11 32 30 10 15 20 16
65 9 26 16 14
73 16 32 20 14
+ 150 82 80 19 12 46 30 21 26 18 20
81 11 26 36 20
78 7 19 21 21
+ 500 81 77 8 8 22 25 19 20 11 15
76 7 30 22 21
74 8 23 19 12
+ 1500 0* 0 0* 0 23 31 20* 19 13* 13
0* 0* 34 15* 9*
0* 0* 35 22* 18*
+ 5000 NT NT 19 21 NT NT
23
22
Positive controls ENNG ENNG ENNG 4NQO 9AA
Concentration (ug/plate) 3 5 2 0.2 80
without S9 674 710 1085 1096 672 955 214 214 2274 2171
712 1110 1070 216 2205
743 1092 1123 212 2035
Positive controls 2AA 2AA 2AA BP 2AA
Concentration (ug/plate) 1 2 10 5 2
With S9 1620 1671 302 300 647 625 312 316 651 569
1598 291 633 316 736
1794 306 594 320 320

Table 2. Main test

Main test with and without S9
  Number of revertants (mean number of colonies per plate)
S9 Mix Test substance concentration (ug/plate) TA100 Mean TA1535 Mean WP2uvrA- Mean TA98 Mean TA1537 Mean
- 0 80 84 9 13 32 30 15 16 21 12
90 14 30 17 5
81 15 27 16 9
- 5 75 73 N/T NT N/T N/T
68
76
- 15 82 78 16 13 NT 25 21 17 15
66 9 17 10
85 15 22 19
- 50 95 85 15 12 41 32 17 21 10 12
89 11 27 22 11
71 9 29 24 16
- 150 95 91 20 18 22 25 25 20 16 16
85 15 19 19 20
94 19 34 16 13
- 500 95 95 16* 17 22 28 24 17 7 9
99 17* 38 11 14
92 19* 24 17 7
- 1500 0* 0 0* 0 21 20 0* 0 0* 0
0* 0* 16 0* 0*
0* 0* 24 0* 0*
- 5000 N/T N/T 11* 13 N/T N/T
10*
18*
                       
                       
+ 0 74 87 11 11 47 42 15   26 25
96 11 39 17 28
92 10 41 22 21
+ 15 56 64 7 11 N/T 18   23 19
56 14 22 17
79 11 13 17
+ 50 68 70 9 10 37 39 14   19 17
62 7 39 22 19
79 13 40 17 13
+ 150 61 68 5 7 30 35 19   9 15
71 7 39 20 21
71 9 37 9 15
+ 500 73 71 7 8 29 31 24   17 19
66 6 31 27 22
75 10 34 15 18
+ 1500 0* 0 0* 0 25 30 5*   21* 20
0* 0* 31 13* 18*
0* 0* 33 8* 20*
+ 5000 N/T N/T 19* 21 N/T N/T
19*
25*
                       
Positive controls ENNG ENNG ENNG 4NQO 9AA
Concentration (ug/plate) 3 5 2 0.2 80
without S9 463 458 244 252 887 891 196 177 1936 1896
473 215 900 162 1633
437 302 887 173 2119
Positive controls 2AA 2AA 2AA BP 2AA
Concentration (ug/plate) 1 2 10 5 2
With S9 2596 2510 305 285 418 416 206 223 458 434
2502 288 365 221 385
2433 262 464 241 458

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, the test substance is considered to be non-mutagenic.
Executive summary:

The study was performed to the requirements of guideline OECD 471 under GLP conditions, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA in both the presence and absence of S-9 mix. Salmonella typhimurium strains TA1535, TA1537, TA98 and TAIOO and Escherichiacoli strain WP2uvrK were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 1.5 and 5000 µg/plate, depending on strain type. The experiment was repeated on a separate day using a similar dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels (1.5, 5 and 15 µg/plate) were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies withinthe normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains, initially at and above 500 µg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate or its toxic limit depending on strain type. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

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