Registration Dossier
Registration Dossier
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EC number: 942-422-6 | CAS number: -
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015
- 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:
- 2 015
- Report date:
- 2015
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
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: March 2014; signature: May 2014
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Reaction products of ((5E)-5-ethylidenebicyclo[2.2.1]hept-2-ene and (5Z)-5-ethylidenebicyclo[2.2.1]hept-2-ene) and 2-methyl-1,3-butadiene, epoxidized
- EC Number:
- 942-422-6
- Molecular formula:
- Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
- IUPAC Name:
- Reaction products of ((5E)-5-ethylidenebicyclo[2.2.1]hept-2-ene and (5Z)-5-ethylidenebicyclo[2.2.1]hept-2-ene) and 2-methyl-1,3-butadiene, epoxidized
- Test material form:
- liquid
- Details on test material:
- - Physical state: Liquid.
- Storage condition of test material: Room temperature in the dark under nitrogen
- Other: Colourless
Constituent 1
Method
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 - Range Finding Test (plate incorporation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method):
Salmonella strains with and without S9-mix - TA100, TA1535, TA1537: 0, 1.5, 5, 15, 50, 150, 500 µg/plate
Salmonella strains with S9 mix - TA98: 0, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
Salmonella strain without S9-mix - TA98: 0, 1.5, 5, 15, 50, 150, 500 µg/plate
E.coli strain with and without S9-mix - WP2uvrA: 0, 15, 50, 150, 500, 1500 µg/plate
One bacterial strain (Ecoli - WP2uvrA dosed in the absence of S9-mix) exhibited excessive toxicity and required a repeat experiment using the following dose range: 0, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Up to seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology. - 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 therefore selected as the vehicle.
- Other: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No correction was made for purity as the test item is a UVCB. All formulations were used within four hours of preparation and were assumed to be stable for this period. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
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 (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)
DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Experiment 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 Subsequently, the procedure for incubation and duration was the same as in Experiment 1. Several manual counts were required, predominantly due to interference in the base agar e.g. minor precipitation of salts/dust particles and marks on the base of the plates slightly distorting the counts.
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - 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:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
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) |
94 69 82 |
(82) 12.5# |
12 20 11 |
(14) 4.9 |
17 28 28 |
(24) 6.4 |
25 21 29 |
(25) 4.0 |
12 13 9 |
(11) 2.1 |
|
1.5 µg |
87 82 80 |
(83) 3.6 |
19 15 7 |
(14) 6.1 |
24 17 12 |
(18) 6.0 |
17 21 19 |
(19) 2.0 |
8 5 12 |
(8) 3.5 |
|
5 µg |
91 69 79 |
(80) 11.0 |
11 9 15 |
(12) 3.1 |
17 13 20 |
(17) 3.5 |
20 25 15 |
(20) 5.0 |
5 19 9 |
(11) 7.2 |
|
15 µg |
91 104 80 |
(92) 12.0 |
17 17 20 |
(18) 1.7 |
19 8 20 |
(16) 6.7 |
19 15 23 |
(19) 4.0 |
13 13 9 |
(12) 2.3 |
|
50 µg |
92 83 71 |
(82) 10.5 |
12 12 12 |
(12) 0.0 |
16 20 19 |
(18) 2.1 |
19 24 21 |
(21) 2.5 |
8 8 11 |
(9) 1.7 |
|
150 µg |
72 64 87 |
(74) 11.7 |
16 9 7 |
(11) 4.7 |
25 25 20 |
(23) 2.9 |
15 15 20 |
(17) 2.9 |
3 12 5 |
(7) 4.7 |
|
500 µg |
12 S 24 S 17 S |
(18) 6.0 |
0 V 0 V 0 V |
(0) 0.0 |
28 17 19 |
(21) 5.9 |
15 S 13 S 13 S |
(14) 1.2 |
0 V 0 V 0 V |
(0) 0.0 |
|
1500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
17 15 21 |
(18) 3.1 |
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 |
21 9 21 |
(17) 6.9 |
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 |
|||||||
505 613 470 |
(529) 74.5 |
176 176 170 |
(174) 3.5 |
106 147 95 |
(116) 27.4 |
172 146 124 |
(147) 24.0 |
2401 2685 1867 |
(2318) 415.3 |
||
|
|||||||||||
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 79 104 |
(94) 13.4# |
9 12 9 |
(10) 1.7 |
32 37 20 |
(30) 8.7 |
28 24 20 |
(24) 4.0 |
21 12 16 |
(16) 4.5 |
|
1.5 µg |
96 95 99 |
(97) 2.1 |
9 17 13 |
(13) 4.0 |
27 17 17 |
(20) 5.8 |
16 17 31 |
(21) 8.4 |
20 15 20 |
(18) 2.9 |
|
5 µg |
111 99 76 |
(95) 17.8 |
13 13 12 |
(13) 0.6 |
28 24 35 |
(29) 5.6 |
25 21 17 |
(21) 4.0 |
23 19 16 |
(19) 3.5 |
|
15 µg |
87 115 102 |
(101) 14.0 |
12 9 21 |
(14) 6.2 |
24 31 29 |
(28) 3.6 |
13 17 20 |
(17) 3.5 |
19 21 12 |
(17) 4.7 |
|
50 µg |
91 82 86 |
(86) 4.5 |
12 12 7 |
(10) 2.9 |
35 33 40 |
(36) 3.6 |
17 23 23 |
(21) 3.5 |
16 9 7 |
(11) 4.7 |
|
150 µg |
78 108 67 |
(84) 21.2 |
9 13 21 |
(14) 6.1 |
31 21 20 |
(24) 6.1 |
21 19 25 |
(22) 3.1 |
9 11 12 |
(11) 1.5 |
|
500 µg |
56 S 57 S 49 S |
(54) 4.4 |
11 S 11 S 9 S |
(10) 1.2 |
31 27 8 |
(22) 12.3 |
25 25 19 |
(23) 3.5 |
8 S 11 S 16 S |
(12) 4.0 |
|
1500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
13 23 21 |
(19) 5.3 |
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 |
11 11 16 |
(13) 2.9 |
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 |
|||||||
2087 1918 1703 |
(1903) 192.5 |
175 196 204 |
(192) 15.0 |
221 182 230 |
(211) 25.5 |
180 235 204 |
(206) 27.6 |
251 358 362 |
(324) 63.0 |
||
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) |
71 88 86 |
(82) 9.3# |
11 15 17 |
(14) 3.1 |
21 27 15 |
(21) 6.0 |
8 23 9 |
(13) 8.4 |
17 16 16 |
(16) 0.6 |
||
0.5 µg |
79 82 83 |
(81) 2.1 |
13 19 9 |
(14) 5.0 |
N/T |
20 13 21 |
(18) 4.4 |
15 20 16 |
(17) 2.6 |
|||
1.5 µg |
78 75 92 |
(82) 9.1 |
24 13 12 |
(16) 6.7 |
16 27 13 |
(19) 7.4 |
13 11 19 |
(14) 4.2 |
13 21 20 |
(18) 4.4 |
||
5 µg |
67 74 95 |
(79) 14.6 |
21 8 8 |
(12) 7.5 |
16 24 31 |
(24) 7.5 |
20 15 9 |
(15) 5.5 |
20 17 17 |
(18) 1.7 |
||
15 µg |
84 75 74 |
(78) 5.5 |
13 16 17 |
(15) 2.1 |
16 24 13 |
(18) 5.7 |
11 15 12 |
(13) 2.1 |
16 11 12 |
(13) 2.6 |
||
50 µg |
88 75 74 |
(79) 7.8 |
12 11 14 |
(12) 1.5 |
19 23 27 |
(23) 4.0 |
8 8 11 |
(9) 1.7 |
8 8 12 |
(9) 2.3 |
||
150 µg |
94 S 69 S 79 S |
(81) 12.6 |
11 S 13 S 11 S |
(12) 1.2 |
15 12 16 |
(14) 2.1 |
9 15 15 |
(13) 3.5 |
0 V 0 V 0 V |
(0) 0.0 |
||
500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
20 S 20 S 20 S |
(20) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
1500 µg |
N/T |
N/T |
23 S 19 S 15 S |
(19) 4.0 |
N/T |
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 |
||||||||
601 552 611 |
(588) 31.6 |
1588 1009 611 |
(1069) 491.3 |
1036 619 687 |
(781) 223.7 |
204 203 210 |
(206) 3.8 |
731 552 528 |
(604) 110.9 |
|||
|
||||||||||||
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) |
64 69 84 |
(72) 10.4# |
13 7 16 |
(12) 4.6 |
21 29 35 |
(28) 7.0 |
12 7 25 |
(15) 9.3 |
23 11 4 |
(13) 9.6 |
||
0.5 µg |
83 74 78 |
(78) 4.5 |
12 20 11 |
(14) 4.9 |
N/T |
N/T |
11 12 8 |
(10) 2.1 |
||||
1.5 µg |
112 68 86 |
(89) 22.1 |
12 16 7 |
(12) 4.5 |
N/T |
18 13 11 |
(14) 3.6 |
9 9 20 |
(13) 6.4 |
|||
5 µg |
100 83 75 |
(86) 12.8 |
7 9 9 |
(8) 1.2 |
N/T |
16 15 20 |
(17) 2.6 |
16 12 12 |
(13) 2.3 |
|||
15 µg |
87 72 80 |
(80) 7.5 |
8 8 9 |
(8) 0.6 |
39 31 32 |
(34) 4.4 |
25 16 20 |
(20) 4.5 |
12 8 4 |
(8) 4.0 |
||
50 µg |
103 78 72 |
(84) 16.4 |
9 10 12 |
(10) 1.5 |
29 32 24 |
(28) 4.0 |
19 15 19 |
(18) 2.3 |
13 5 12 |
(10) 4.4 |
||
150 µg |
99 72 110 |
(94) 19.6 |
9 8 8 |
(8) 0.6 |
23 15 20 |
(19) 4.0 |
9 9 15 |
(11) 3.5 |
5 23 7 |
(12) 9.9 |
||
500 µg |
79 S 65 S 60 S |
(68) 9.8 |
9 S 12 S 9 S |
(10) 1.7 |
15 21 17 |
(18) 3.1 |
31 13 17 |
(20) 9.5 |
9 S 1 S 4 S |
(5) 4.0 |
||
1500 µg |
N/T |
N/T |
24 17 19 |
(20) 3.6 |
0 V 0 V 0 V |
(0) 0.0 |
N/T |
|||||
5000 µg |
N/T |
N/T |
16 S 28 S 21 S |
(22) 6.0 |
N/T |
N/T |
||||||
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 |
||||||||
964 981 1028 |
(991) 33.2 |
139 166 174 |
(160) 18.3 |
103 99 123 |
(108) 12.9 |
79 114 124 |
(106) 23.6 |
72 128 131 |
(110) 33.2 |
|||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
P: Test item precipitate
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
T: Toxic, no bacterial background lawn
#: Standard deviation
N/T: Not tested at this dose level
Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
124 |
|
24 |
|
37 |
|
25 |
|
5 |
|
80 |
(94) |
25 |
(23) |
24 |
(29) |
20 |
(23) |
4 |
(6) |
78 |
|
20 |
|
27 |
|
25 |
|
9 |
|
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
112 |
|
8 |
|
32 |
|
21 |
|
12 |
|
99 |
(106) |
9 |
(13) |
37 |
(31) |
25 |
(19) |
8 |
(12) |
108 |
|
23 |
|
23 |
|
11 |
|
16 |
|
|
15 |
|
|
||||||
28 |
(22)† |
||||||||
24 |
|
||||||||
† Experimental procedure repeated at a later date (without S9-mix) due to toxicity in the original test.
All positive and vehicle and negative controls were within laboratory historic values.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results:
negative
Under the conditions of this study the test item 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 and Japanese guidelines for bacterial mutagenicity testing under GLP, 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 WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using both the Ames plate incorporation and pre incubation methods 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 dose range was amended following the results of Experiment 1 and ranged between 0.5 and 5000 µg/plate depending on bacterial tester strain and absence or presence of S9-mix. Up to seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology. 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 sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (light and globular in appearance) was noted under an inverted microscope at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies. It was concluded that, under the conditions of this assay, the test substance 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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