Registration Dossier
Registration Dossier
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EC number: 915-782-7 | CAS number: -
Genetic toxicity: in vitro
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:
- 25-07-2018 to 06-08-2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
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
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: July 2017; signature: November 2017
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran
- EC Number:
- 915-782-7
- Molecular formula:
- C12H14O
- IUPAC Name:
- Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran
- Test material form:
- liquid
- Details on test material:
- - Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, under Nitrogen, in the dark
- Other: Colourless (sponsor) ; clear colourless (laboratory)
Constituent 1
Method
- Target gene:
- histidine or tryptophan locus
Species / strainopen allclose all
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- 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:
- Experiment 1 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 0.5, 1.5, 5, 15, 50, 150, 500 and/or 1500 µg/plate (depending on toxicity).
Eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item. The dose levels were selected based on the results of Experiment 1. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item 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. Dimethyl sulphoxide was selected as the vehicle.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- 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: Experiment 1. in medium; in agar (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation)
DURATION
- Exposure duration:
Experiment 1 and 2: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media All of the plates were sealed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure (37 ± 3 ºC for approximately 48 hours) to minimize potential losses of the test item from the plates. After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Rationale for test conditions:
- Conditions were selected in accordance with the relevant guidelines.
- Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal. - Statistics:
- Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).
Results and discussion
Test resultsopen allclose all
- 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:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
Any other information on results incl. tables
Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
100 122 106 |
(109) 11.4# |
20 20 15 |
(18) 2.9 |
31 19 31 |
(27) 6.9 |
25 17 18 |
(20) 4.4 |
8 5 13 |
(9) 4.0 |
|
1.5 µg |
125 98 117 |
(113) 13.9 |
18 17 12 |
(16) 3.2 |
29 30 27 |
(29) 1.5 |
25 25 18 |
(23) 4.0 |
16 9 15 |
(13) 3.8 |
|
5 µg |
125 105 93 |
(108) 16.2 |
25 22 16 |
(21) 4.6 |
29 24 37 |
(30) 6.6 |
28 19 25 |
(24) 4.6 |
12 12 10 |
(11) 1.2 |
|
15 µg |
109 90 92 |
(97) 10.4 |
16 22 18 |
(19) 3.1 |
35 27 29 |
(30) 4.2 |
30 29 35 |
(31) 3.2 |
10 15 7 |
(11) 4.0 |
|
50 µg |
101 90 100 |
(97) 6.1 |
23 18 22 |
(21) 2.6 |
27 33 32 |
(31) 3.2 |
25 27 12 |
(21) 8.1 |
13 14 6 |
(11) 4.4 |
|
150 µg |
89 S 82 S 105 S |
(92) 11.8 |
18 12 20 |
(17) 4.2 |
29 34 30 |
(31) 2.6 |
23 30 26 |
(26) 3.5 |
2 13 7 |
(7) 5.5 |
|
500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
16 S 25 S 22 S |
(21) 4.6 |
0 T 0 T 0 T |
(0) 0.0 |
|
1500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
20 S 21 S 24 S |
(22) 2.1 |
0 T 0 T 0 T |
(0) 0.0 |
|
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
|
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
|||||||
851 813 806 |
(823) 24.2 |
1227 1387 1471 |
(1362) 124.0 |
1301 1244 1198 |
(1248) 51.6 |
241 261 262 |
(255) 11.8 |
617 368 316 |
(434) 160.9 |
||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
121 118 119 |
(119) 1.5# |
12 13 11 |
(12) 1.0 |
37 34 56 |
(42) 11.9 |
16 23 23 |
(21) 4.0 |
8 15 16 |
(13) 4.4 |
|
1.5 µg |
100 96 119 |
(105) 12.3 |
10 13 11 |
(11) 1.5 |
38 38 35 |
(37) 1.7 |
33 28 32 |
(31) 2.6 |
22 12 11 |
(15) 6.1 |
|
5 µg |
131 134 120 |
(128) 7.4 |
12 15 17 |
(15) 2.5 |
35 44 40 |
(40) 4.5 |
22 33 29 |
(28) 5.6 |
16 12 19 |
(16) 3.5 |
|
15 µg |
135 120 105 |
(120) 15.0 |
14 7 19 |
(13) 6.0 |
28 42 29 |
(33) 7.8 |
24 35 24 |
(28) 6.4 |
8 14 17 |
(13) 4.6 |
|
50 µg |
111 116 142 |
(123) 16.6 |
12 24 8 |
(15) 8.3 |
32 40 34 |
(35) 4.2 |
18 24 34 |
(25) 8.1 |
9 10 15 |
(11) 3.2 |
|
150 µg |
135 119 118 |
(124) 9.5 |
9 11 10 |
(10) 1.0 |
44 40 46 |
(43) 3.1 |
28 19 34 |
(27) 7.5 |
12 9 13 |
(11) 2.1 |
|
500 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
30 S 21 S 27 S |
(26) 4.6 |
17 23 24 |
(21) 3.8 |
6 S 6 S 10 S |
(7) 2.3 |
|
1500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
|
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
|
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
|||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
|||||||
988 1537 1823 |
(1449) 424.3 |
308 264 331 |
(301) 34.0 |
284 295 292 |
(290) 5.7 |
112 119 132 |
(121) 10.1 |
213 207 217 |
(212) 5.0 |
||
Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
90 111 104 |
(102) 10.7# |
17 16 21 |
(18) 2.6 |
20 23 19 |
(21) 2.1 |
19 23 17 |
(20) 3.1 |
6 12 15 |
(11) 4.6 |
|
0.15 µg |
127 117 108 |
(117) 9.5 |
33 20 25 |
(26) 6.6 |
16 16 24 |
(19) 4.6 |
N/T |
15 13 19 |
(16) 3.1 |
||
0.5 µg |
97 107 109 |
(104) 6.4 |
32 17 15 |
(21) 9.3 |
33 27 24 |
(28) 4.6 |
24 27 10 |
(20) 9.1 |
13 19 15 |
(16) 3.1 |
|
1.5 µg |
107 112 93 |
(104) 9.8 |
11 22 22 |
(18) 6.4 |
19 14 15 |
(16) 2.6 |
25 18 16 |
(20) 4.7 |
14 16 12 |
(14) 2.0 |
|
5 µg |
90 113 121 |
(108) 16.1 |
28 21 8 |
(19) 10.1 |
19 34 38 |
(30) 10.0 |
21 10 22 |
(18) 6.7 |
13 14 16 |
(14) 1.5 |
|
15 µg |
126 85 82 |
(98) 24.6 |
24 18 32 |
(25) 7.0 |
29 32 24 |
(28) 4.0 |
21 24 14 |
(20) 5.1 |
21 15 10 |
(15) 5.5 |
|
50 µg |
93 117 90 |
(100) 14.8 |
29 29 14 |
(24) 8.7 |
22 12 23 |
(19) 6.1 |
32 13 20 |
(22) 9.6 |
10 10 17 |
(12) 4.0 |
|
150 µg |
105 S 110 S 97 S |
(104) 6.6 |
20 24 22 |
(22) 2.0 |
25 S 26 S 20 S |
(24) 3.2 |
15 21 14 |
(17) 3.8 |
5 6 6 |
(6) 0.6 |
|
500 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
20 S 12 S 12 S |
(15) 4.6 |
3 S 6 S 5 S |
(5) 1.5 |
|
1500 µg |
N/T |
N/T |
N/T |
0 V 0 V 0 V |
(0) 0.0 |
N/T |
|||||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
|||||||
595 617 621 |
(611) 14.0 |
232 215 219 |
(222) 8.9 |
626 597 492 |
(572) 70.5 |
213 218 209 |
(213) 4.5 |
103 86 100 |
(96) 9.1 |
||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
138 105 135 |
(126) 18.2# |
14 11 10 |
(12) 2.1 |
36 31 32 |
(33) 2.6 |
26 24 28 |
(26) 2.0 |
16 26 21 |
(21) 5.0 |
|
0.15 µg |
124 130 95 |
(116) 18.7 |
20 15 12 |
(16) 4.0 |
N/T |
N/T |
N/T |
||||
0.5 µg |
119 105 121 |
(115) 8.7 |
14 21 10 |
(15) 5.6 |
34 27 29 |
(30) 3.6 |
29 37 35 |
(34) 4.2 |
21 16 14 |
(17) 3.6 |
|
1.5 µg |
92 112 97 |
(100) 10.4 |
17 13 21 |
(17) 4.0 |
29 36 34 |
(33) 3.6 |
20 22 39 |
(27) 10.4 |
19 18 15 |
(17) 2.1 |
|
5 µg |
124 115 126 |
(122) 5.9 |
25 18 14 |
(19) 5.6 |
37 33 31 |
(34) 3.1 |
17 21 36 |
(25) 10.0 |
15 15 12 |
(14) 1.7 |
|
15 µg |
105 95 90 |
(97) 7.6 |
17 18 14 |
(16) 2.1 |
30 24 31 |
(28) 3.8 |
22 28 24 |
(25) 3.1 |
22 11 18 |
(17) 5.6 |
|
50 µg |
95 118 115 |
(109) 12.5 |
13 11 9 |
(11) 2.0 |
28 23 28 |
(26) 2.9 |
23 28 21 |
(24) 3.6 |
26 17 20 |
(21) 4.6 |
|
150 µg |
108 128 138 |
(125) 15.3 |
11 14 14 |
(13) 1.7 |
26 39 25 |
(30) 7.8 |
16 24 24 |
(21) 4.6 |
14 23 18 |
(18) 4.5 |
|
500 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
31 S 20 S 22 S |
(24) 5.9 |
21 25 25 |
(24) 2.3 |
6 S 7 S 8 S |
(7) 1.0 |
|
1500 µg |
N/T |
N/T |
0 V 0 V 0 V |
(0) 0.0 |
24 S 21 S 12 S |
(19) 6.2 |
0 V 0 V 0 V |
(0) 0.0 |
|||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
|||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
|||||||
1518 1380 1502 |
(1467) 75.5 |
252 250 259 |
(254) 4.7 |
183 144 149 |
(159) 21.2 |
101 120 135 |
(119) 17.0 |
231 231 223 |
(228) 4.6 |
||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
N/T: Not tested at this dose level
S: Sparse bacterial background lawn
T: Toxic, no bacterial background lawn
V: Very weak bacterial background lawn
#: Standard deviation
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study the test material was considered to be non-mutagenic in the presence and absence of S9 activation. - Executive summary:
The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100, ICH S2(R1) and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item using both the Ames pre incubation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations.The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was determined by the results of experiment 1 and ranged between 0.15 and 1500 µg/plate depending on strain type and/or exposure to S9 mix. Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.There was a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 150 and 500 µg/plate in the absence and presence of metabolic activation (S9-mix), respectively. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. A minor statistical value was noted in Experiment 1 (TA98 at 15 µg/plate in the absence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.No biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. A minor statistical value was noted in Experiment 2 (TA1535 at 5 µg/plate in the presence of S9-mix), however this response was within the in house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance. It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.
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