Registration Dossier
Registration Dossier
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EC number: 947-375-5 | 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:
- 19 July - 28 August 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD Guideline 471 without any deviation
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- dated 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- dated 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- August 1998
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 28 October 2016
- Type of assay:
- bacterial reverse mutation assay
Test material
- Test material form:
- solid
- Details on test material:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: K17 039-1
- Production date of the batch: February 2017
- Expiration date of the lot/batch: 15 February 2019
- Storage conditions: room temperature
- Appearance: dark brown solid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: K17 039-1
- Production date of the batch: February 2017
- Expiration date of the lot/batch: 15 February 2019
- Purity test date: February 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in pre-dried tetrahydrofuran by mixing on a vortex mixer and sonication for 5 minutes at approximately 40°C on the day of each experiment. No correction was required for purity. Tetrahydrofuran is toxic to the bacterial cells at and above 50 µL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 µL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). All formulations were used within four hours of preparation and were assumed to be stable for this period.
Method
- Target gene:
- histidine locus for Salmonella strains and tryptophan for E. coli strain
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
- Test concentrations with justification for top dose:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in tetrahydrofuran at 200 mg/mL in solubility checks performed in house. Tetrahydrofuran was therefore selected as the vehicle.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- untreated
- 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
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from:
- University of California, Berkeley, on culture discs, on 04 August 1995
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 20 minutes in Experiment 2.
- Exposure duration: ca. 48 hours
CONTROLS:
- Vehicle/solvent control: Tetrahydrofuran; performed in triplicate
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate; performed in triplicate
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation; performed in triplicate
- Sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).
NUMBER OF REPLICATIONS: Triplicate
- OTHER: All of the plates were incubated at 37 ± 3 °C 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). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count. - Rationale for test conditions:
- The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate (i.e. maximum recommended dose level). 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 was 15 to 5000 µg/plate. Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
- Evaluation criteria:
- Criteria for determining a positive result:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- 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 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 making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- None
Results and discussion
Test results
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (greasy in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
MUTAGENICITY
- The vehicle (tetrahydrofuran) 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.
- No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
- No significant 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 1 (plate incorporation method) and Experiment 2 (pre incubation method).
- Refer Tables 7.6.1/1 to 7.6.1/5 for more details.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Refer Table 7.6.1/6
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/6
OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are presented in 7.6.1/1 and were considered to be 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 7.6.1/1: Spontaneous Mutation Rates (Concurrent Negative Controls)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
Experiment 1 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
82 |
|
28 |
|
21 |
|
26 |
|
8 |
|
104 |
(97) |
11 |
(22) |
32 |
(24) |
21 |
(23) |
9 |
(12) |
104 |
|
26 |
|
19 |
|
23 |
|
18 |
|
Experiment 2 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
83 |
|
31 |
|
33 |
|
21 |
|
7 |
|
64 |
(77) |
25 |
(25) |
28 |
(33) |
22 |
(21) |
10 |
(11) |
84 |
|
19 |
|
37 |
|
19 |
|
15 |
|
Table 7.6.1/2: Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 09 August 2017 |
To: 12 August 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (THF) |
127 110 114 |
(117) 8.9# |
28 20 24 |
(24) 4.0 |
20 20 25 |
(22) 2.9 |
32 27 21 |
(27) 5.5 |
11 8 14 |
(11) 3.0 |
||
1.5 µg |
125 101 99 |
(108) 14.5 |
25 13 21 |
(20) 6.1 |
18 28 23 |
(23) 5.0 |
38 27 21 |
(29) 8.6 |
7 13 17 |
(12) 5.0 |
||
5 µg |
111 99 94 |
(101) 8.7 |
26 26 26 |
(26) 0.0 |
16 22 23 |
(20) 3.8 |
27 27 24 |
(26) 1.7 |
13 13 17 |
(14) 2.3 |
||
15 µg |
97 92 123 |
(104) 16.6 |
25 28 24 |
(26) 2.1 |
17 26 15 |
(19) 5.9 |
17 35 17 |
(23) 10.4 |
9 13 15 |
(12) 3.1 |
||
50 µg |
106 127 124 |
(119) 11.4 |
21 19 19 |
(20) 1.2 |
25 22 21 |
(23) 2.1 |
23 17 23 |
(21) 3.5 |
13 13 8 |
(11) 2.9 |
||
150 µg |
125 125 110 |
(120) 8.7 |
27 26 20 |
(24) 3.8 |
31 17 24 |
(24) 7.0 |
17 24 24 |
(22) 4.0 |
13 13 12 |
(13) 0.6 |
||
500 µg |
125 106 126 |
(119) 11.3 |
14 26 27 |
(22) 7.2 |
31 14 26 |
(24) 8.7 |
25 24 20 |
(23) 2.6 |
13 20 6 |
(13) 7.0 |
||
1500 µg |
115 P 132 P 125 P |
(124) 8.5 |
19 P 27 P 21 P |
(22) 4.2 |
12 P 12 P 16 P |
(13) 2.3 |
28 P 33 P 24 P |
(28) 4.5 |
16 P 7 P 14 P |
(12) 4.7 |
||
5000 µg |
114 P 110 P 99 P |
(108) 7.8 |
17 P 13 P 15 P |
(15) 2.0 |
23 P 27 P 23 P |
(24) 2.3 |
21 P 19 P 24 P |
(21) 2.5 |
15 P 9 P 13 P |
(12) 3.1 |
||
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 |
||||||||
775 722 618 |
(705) 79.9 |
559 450 677 |
(562) 113.5 |
838 805 833 |
(825) 17.8 |
271 266 288 |
(275) 11.5 |
272 144 200 |
(205) 64.2 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
P: Test item precipitate
#: Standard deviation
Table 7.6.1/3: Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 09 August 2017 |
To: 12 August 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (THF) |
115 96 123 |
(111) 13.9# |
21 26 29 |
(25) 4.0 |
34 25 27 |
(29) 4.7 |
25 38 30 |
(31) 6.6 |
13 21 13 |
(16) 4.6 |
||
1.5 µg |
72 115 115 |
(101) 24.8 |
21 26 18 |
(22) 4.0 |
31 20 25 |
(25) 5.5 |
38 37 27 |
(34) 6.1 |
13 12 18 |
(14) 3.2 |
||
5 µg |
117 96 109 |
(107) 10.6 |
28 21 31 |
(27) 5.1 |
30 32 23 |
(28) 4.7 |
39 28 29 |
(32) 6.1 |
10 9 17 |
(12) 4.4 |
||
15 µg |
117 119 84 |
(107) 19.7 |
23 21 19 |
(21) 2.0 |
21 30 18 |
(23) 6.2 |
30 17 32 |
(26) 8.1 |
11 19 22 |
(17) 5.7 |
||
50 µg |
90 106 105 |
(100) 9.0 |
11 30 26 |
(22) 10.0 |
21 22 25 |
(23) 2.1 |
21 23 26 |
(23) 2.5 |
11 11 14 |
(12) 1.7 |
||
150 µg |
115 112 116 |
(114) 2.1 |
14 30 24 |
(23) 8.1 |
24 21 29 |
(25) 4.0 |
27 22 23 |
(24) 2.6 |
15 17 14 |
(15) 1.5 |
||
500 µg |
113 137 113 |
(121) 13.9 |
19 31 28 |
(26) 6.2 |
25 27 26 |
(26) 1.0 |
26 40 33 |
(33) 7.0 |
10 18 18 |
(15) 4.6 |
||
1500 µg |
121 P 130 P 119 P |
(123) 5.9 |
27 P 20 P 30 P |
(26) 5.1 |
33 P 37 P 28 P |
(33) 4.5 |
21 P 28 P 37 P |
(29) 8.0 |
12 P 18 P 17 P |
(16) 3.2 |
||
5000 µg |
119 P 113 P 118 P |
(117) 3.2 |
28 P 24 P 31 P |
(28) 3.5 |
37 P 27 P 28 P |
(31) 5.5 |
37 P 20 P 26 P |
(28) 8.6 |
21 P 13 P 21 P |
(18) 4.6 |
||
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 |
||||||||
1137 1032 1049 |
(1073) 56.4 |
260 262 230 |
(251) 17.9 |
155 143 132 |
(143) 11.5 |
221 215 196 |
(211) 13.1 |
373 480 413 |
(422) 54.1 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
P: Test item precipitate
#: Standard deviation
Table 7.6.1/4: Test Results: Experiment 2 – Without Metabolic Activation
Test Period |
From: 24 August 2017 |
To: 27 August 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (THF) |
72 61 73 |
(69) 6.7# |
25 16 16 |
(19) 5.2 |
40 34 32 |
(35) 4.2 |
23 25 23 |
(24) 1.2 |
11 13 12 |
(12) 1.0 |
||
15 µg |
69 60 56 |
(62) 6.7 |
22 23 21 |
(22) 1.0 |
24 23 39 |
(29) 9.0 |
18 26 22 |
(22) 4.0 |
9 7 10 |
(9) 1.5 |
||
50 µg |
70 49 65 |
(61) 11.0 |
15 15 26 |
(19) 6.4 |
26 39 32 |
(32) 6.5 |
24 22 29 |
(25) 3.6 |
13 13 15 |
(14) 1.2 |
||
150 µg |
64 64 61 |
(63) 1.7 |
19 14 21 |
(18) 3.6 |
29 30 26 |
(28) 2.1 |
20 13 20 |
(18) 4.0 |
11 17 11 |
(13) 3.5 |
||
500 µg |
66 47 60 |
(58) 9.7 |
15 16 17 |
(16) 1.0 |
30 37 29 |
(32) 4.4 |
11 22 21 |
(18) 6.1 |
9 21 13 |
(14) 6.1 |
||
1500 µg |
75 P 78 P 85 P |
(79) 5.1 |
25 P 17 P 22 P |
(21) 4.0 |
26 P 26 P 36 P |
(29) 5.8 |
23 P 21 P 23 P |
(22) 1.2 |
16 P 12 P 15 P |
(14) 2.1 |
||
5000 µg |
84 P 85 P 76 P |
(82) 4.9 |
19 P 17 P 16 P |
(17) 1.5 |
34 P 32 P 32 P |
(33) 1.2 |
23 P 22 P 24 P |
(23) 1.0 |
12 P 12 P 16 P |
(13) 2.3 |
||
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 |
||||||||
1635 1511 2198 |
(1781) 366.1 |
278 306 360 |
(315) 41.7 |
1112 1182 959 |
(1084) 114.0 |
291 308 285 |
(295) 11.9 |
275 344 356 |
(325) 43.7 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
P: Test item precipitate
#: Standard deviation
Table 7.6.1/5: Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 24 August 2017 |
To: 27 August 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (THF) |
61 74 60 |
(65) 7.8# |
21 21 21 |
(21) 0.0 |
38 33 44 |
(38) 5.5 |
33 15 20 |
(23) 9.3 |
11 10 11 |
(11) 0.6 |
||
15 µg |
59 69 54 |
(61) 7.6 |
34 27 22 |
(28) 6.0 |
29 41 33 |
(34) 6.1 |
30 28 18 |
(25) 6.4 |
11 19 13 |
(14) 4.2 |
||
50 µg |
64 50 68 |
(61) 9.5 |
27 24 29 |
(27) 2.5 |
34 44 41 |
(40) 5.1 |
30 27 24 |
(27) 3.0 |
21 4 9 |
(11) 8.7 |
||
150 µg |
81 72 56 |
(70) 12.7 |
21 19 24 |
(21) 2.5 |
46 34 45 |
(42) 6.7 |
32 26 26 |
(28) 3.5 |
11 9 14 |
(11) 2.5 |
||
500 µg |
88 73 70 |
(77) 9.6 |
21 22 20 |
(21) 1.0 |
46 45 49 |
(47) 2.1 |
14 24 26 |
(21) 6.4 |
12 10 5 |
(9) 3.6 |
||
1500 µg |
71 P 70 P 70 P |
(70) 0.6 |
22 P 25 P 20 P |
(22) 2.5 |
43 P 45 P 37 P |
(42) 4.2 |
25 P 26 P 16 P |
(22) 5.5 |
11 P 11 P 11 P |
(11) 0.0 |
||
5000 µg |
66 P 66 P 72 P |
(68) 3.5 |
22 P 21 P 21 P |
(21) 0.6 |
39 P 41 P 41 P |
(40) 1.2 |
22 P 21 P 24 P |
(22) 1.5 |
14 P 11 P 15 P |
(13) 2.1 |
||
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 |
||||||||
1160 1121 1217 |
(1166) 48.3 |
944 1041 1119 |
(1035) 87.7 |
206 254 284 |
(248) 39.3 |
183 188 175 |
(182) 6.6 |
419 479 474 |
(457) 33.3 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
P: Test item precipitate
#: Standard deviation
Table 7.6.1/6: History Profile of Vehicle and Positive Control Values
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values† |
274 |
278 |
504 |
285 |
26 |
13 |
461 |
229 |
526 |
299 |
506 |
282 |
42 |
51 |
39 |
49 |
|||||||||||||||||
Min |
60 |
61 |
7 |
7 |
222 |
278 |
10 |
12 |
11 |
10 |
4 |
6 |
87 |
98 |
89 |
93 |
|||||||||||||||||
Max |
166 |
175 |
31 |
29 |
376 |
388 |
58 |
43 |
45 |
46 |
27 |
27 |
237 |
254 |
174 |
177 |
|||||||||||||||||
Mean |
91 |
95 |
16 |
14 |
286 |
333 |
24 |
27 |
21 |
24 |
12 |
13 |
156 |
164 |
123 |
137 |
|||||||||||||||||
SD |
19.3 |
19.1 |
4.5 |
4.0 |
48.7 |
37.6 |
5.6 |
5.9 |
6.2 |
6.1 |
3.8 |
3.4 |
42.2 |
35.6 |
23.1 |
21.2 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2015 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
276 |
280 |
252 |
264 |
13 |
13 |
231 |
227 |
262 |
276 |
253 |
261 |
20 |
35 |
20 |
35 |
|
||||||||||||||||
Min |
222 |
250 |
79 |
118 |
953 |
673 |
116 |
103 |
100 |
78 |
164 |
97 |
430 |
494 |
745 |
325 |
|
||||||||||||||||
Max |
2266 |
2402 |
2779 |
457 |
3140 |
1655 |
2769 |
550 |
502 |
705 |
2318 |
823 |
1696 |
2264 |
3662 |
1174 |
|
||||||||||||||||
Mean |
614 |
927 |
472 |
246 |
2303 |
1093 |
792 |
266 |
222 |
218 |
911 |
336 |
761 |
1461 |
2257 |
569 |
|
||||||||||||||||
SD |
260.6 |
452.5 |
434.8 |
55.7 |
815.2 |
376.5 |
342.1 |
97.7 |
70.2 |
107.6 |
412.4 |
135.7 |
350.0 |
382.0 |
790.7 |
220.3 |
|
||||||||||||||||
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2016 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values |
399 |
401 |
758 |
393 |
60 |
30 |
690 |
345 |
788 |
415 |
762 |
398 |
32 |
32 |
16 |
24 |
|||||||||||||||||
Min |
63 |
66 |
8 |
8 |
216 |
221 |
10 |
13 |
8 |
12 |
3 |
4 |
97 |
104 |
78 |
52 |
|||||||||||||||||
Max |
154 |
156 |
34 |
39 |
340 |
375 |
53 |
53 |
49 |
51 |
24 |
23 |
268 |
243 |
148 |
166 |
|||||||||||||||||
Mean |
90 |
93 |
15 |
15 |
268 |
310 |
22 |
27 |
21 |
25 |
12 |
13 |
161 |
159 |
118 |
110 |
|||||||||||||||||
SD |
14.5 |
14.3 |
4.5 |
5.2 |
26.4 |
31.1 |
5.8 |
6.3 |
4.8 |
5.7 |
3.5 |
3.5 |
39.2 |
32.3 |
17.0 |
29.3 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2016 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
409 |
406 |
381 |
386 |
30 |
28 |
341 |
335 |
388 |
385 |
379 |
381 |
14 |
24 |
8 |
16 |
|
||||||||||||||||
Min |
221 |
284 |
84 |
92 |
897 |
629 |
107 |
102 |
100 |
96 |
95 |
101 |
445 |
574 |
1674 |
372 |
|
||||||||||||||||
Max |
2222 |
2863 |
2994 |
879 |
2326 |
2140 |
1611 |
637 |
449 |
4357 |
1413 |
639 |
1117 |
1855 |
2823 |
945 |
|
||||||||||||||||
Mean |
724 |
1264 |
854 |
240 |
1633 |
950 |
718 |
240 |
186 |
188 |
406 |
290 |
743 |
1271 |
2379 |
535 |
|
||||||||||||||||
SD |
320.4 |
562.9 |
664.9 |
62.1 |
564.5 |
382.7 |
338.6 |
98.2 |
49.8 |
230.8 |
227.0 |
92.7 |
214.6 |
326.5 |
426.2 |
143.3 |
|
||||||||||||||||
Applicant's summary and conclusion
- Conclusions:
- Under the test conditions, the test item is not considered as mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.
The vehicle (tetrahydrofuran) 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.
No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
A test item precipitate (greasy in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant 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 1 (plate incorporation method) and Experiment 2 (pre incubation method).
Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.
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