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EC number: 610-204-7 | CAS number: 446299-90-7
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Genetic toxicity in vitro
Description of key information
In vitro gene mutation study in bacteria: Key study. Test method according to OECD 471, GLP study. The test item did not induce any mutagenic change in the bacterial reverse mutation test in any of the strains tested with and without metabolic activation up to 5000 µg/plate.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03 August 2020 - 01 Octubre 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his D (S. typhimurium TA 98); his C (S. typhimurium TA 1537); his G (S. typhimurium TA 100 and TA1535); tryp E (E. coli WP2 uvrA pKM101)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: ΔuvrB and rfa mutated
- Remarks:
- (TA 98 and TA 100: pKM 101)
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- other: uvrA, pKM 101 mutated
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : S9 fraction prepared from Sprague Dawley rat liver homogenate and provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA).
- method of preparation of S9 mix : 10% S9 fraction, 8 mM MgCL2-6H2O, 33 mM KCl, 5 mM Glucose-6-Phosphate Na2, 4 mM NADP Na2 and 0.1 M Phosphate buffer pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL of S9-mix.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility test: 500 μL of S9-mix were added to 2 mL of top agar maintained at 45ºC, and poured after homogenization on the bottom agar (20 ml) onto a Petri plate (90 mm in diameter) (n = 3). Plates were incubated for 48 - 72 hours at 37°C and then examined. - Test concentrations with justification for top dose:
- Without metabolic activation: 1500, 500, 150, 50 and 15 μg/plate
With metabolic activation: 5, 1.5, 0.5, 0.15 and 0.05 μg/plate
Top doses based on several preliminary cytotoxicity assays (see "Addtional information on results") - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was found soluble in DMSO at the highest tested concentration. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulfoxide (DMSO), acetone, NaCl 0.15M
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-aminoanthracene (1, 2 μg/plate; S. typhimurium strains, + S9), cis-Platinum (II) Diamine Dichloride (1 μg/plate; E.coli, - S9)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
1. Plate incorporation (initial mutation test): A stock solution of the test item was prepared at 100 mg/mL. In a test tube, 0.1 mL of the bacterial suspension containing 1-9 E09 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled at 45ºC containing 10% (v/v) of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains, or containing 5% (v/v) of nutrient broth nº2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL for Escherichia coli strain. In the assay with metabolic activation, the protocol is similar to the described above, except that, 500 μL of S9-mix fraction is quickly added, before pouring the mixture onto the plates. After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate.
2. Pre-incubation (confirmatory mutation test +S9): The test item solution with the test strain, and 500 μL of S9-mix fraction are preincubated with shaking for 30 min., at 37ºC prior to mixing with the overlay agar and pouring onto the minimal agar plate.. After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes (confirmatory mutation test)
- Exposure duration/duration of treatment: 48-72 hours
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Preliminary cytotoxicity test (Strain TA100): In a test tube 0.1 mL of the bacterial suspension (1-9 E03 bacteria /mL) and 0.1 mL of the stock solution and dilutions were successively added to 2 mL of top agar at 45ºC, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 48-72 h at 37ºC, and the colonies counted. A negative control containing the blank alone was run in parallel. In case of bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring.
METHODS FOR MEASUREMENTS OF GENOTOXICIY
In the bacterial reverse mutation test, mutations are detected which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.
After a 48-72-hour incubation period at 37ºC, revertant colonies were manually counted in each plate. The following ratio was calculated per plate: R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item.
- OTHER:
- Sterility test: Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45ºC, and poured after homogenization on the bottom agar (20 mL) onto a Petri plate (90 mm in diameter) (n=3). Plates are incubated for 48-72 hours at 37ºC and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol. - Rationale for test conditions:
- Results of sterility controls show the absence of any bacterial growth in the presence of test item and S9-mix. Concentrations were selected based on the results of the bacteriostatic activity controls. Values and frequency are within the laboratory's historical control ranges.
- Evaluation criteria:
- The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment. - Key result
- Species / strain:
- S. typhimurium TA 98
- 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
- Key result
- Species / strain:
- S. typhimurium 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
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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
- Key result
- 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
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
: None observed.
RANGE-FINDING/SCREENING STUDIES: In the preliminary cytotoxicity test (strain TA100) a high toxicity was found for doses from 1500 to 5000 μg/plate. The doses in the mutagenic assay were selected according to these results. However, in this first mutagenic assay (data not shown) no revertant colonies were counted for all the doses studied in presence of metabolic activation. Evidence of toxicity was demonstrated by the absence or a thinning of the background lawn of non -revertant bacteria, in all strains. In absence of metabolic activation, the toxicity was also observed by the absence or a thinning of the background lawn of non -revertant bacteria, in all strains for the highest doses tested from 3000 to 5000 μg/plate. These results were not consistent with the first cytotoxicity study on TA100. In order to justify acceptable range of doses for the test additional assays were carried out under the same conditions than a mutagenicity test without metabolic activation. Reference substances were not used in this assay. Results of these assays are presented in the tables 4.1, 4.2 and 4.3 below. According to these results, doses were selected for the definitive mutagenic assay (assays 2 and 3 in the table 5 below).
STUDY RESULTS
- Concurrent vehicle negative and positive control data: see tables below.
Ames test:
- Signs of toxicity: see table 5 below.
- Individual plate counts: see table 5 below.
- Mean number of revertant colonies per plate and standard deviation: see table 5 below.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see table 6 below
- Negative (solvent/vehicle) historical control data: see table 6 below
- There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory. - Conclusions:
- The test item did not induce any mutagenic change in the bacterial reverse mutation test in any of the strains tested with and without metabolic activation.
- Executive summary:
A bacterial reverse mutation test was conducted on the test substance according to OECD guideline 471 under GLP conditions. The study was performed in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA, in the absence and presence of metabolic activation. The test substance was found soluble in DMSO which was used as vehicle. The metabolic activation system (S9 fraction) was prepared from Sprague Dawley rat liver homogenate. In the preliminary cytotoxicity test (strain TA100) a high toxicity was found for doses from 1500 to 5000 μg/plate. The doses in the mutagenic assay were selected according to these results. However, in this first mutagenic assay (assay nº1) no revertant colonies were counted for all the doses studied in presence of metabolic activation and toxicity was not consistent with the results of the first cytotoxicity study on TA100. Therefore, additional cytotoxicity assays in all strains were carried out in order to justify acceptable range of doses. According to the results obtained, the second (plate incorporation method with and without S9) and third (plate incorporation method without S9 and pre-incubation method with S9) mutagenic assays were performed with the following doses: 1500, 500, 150, 50 and 15 μg/plate without S9 and 5, 1.5, 0.5, 0.15 and 0.05 μg/plate with S9. Untreated, solvent controls and strain specific positive controls were included in the assays and the values obtained were within ranges of the historical control values of the laboratory in all strains. All validity criteria were fulfilled. The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation. Based on these results, the test item can be considered as not mutagenic according to the OECD TG 471.
Reference
Table 3. Sterility control.
Serie |
Doses |
Colony number/plate |
|||
Control n° 2 |
1 |
2 |
3 |
||
Solution of |
1500 µg /plate |
0 |
0 |
0 |
|
500 µg /plate |
0 |
0 |
0 |
||
T-A12 BATCH: I20196A |
150 µg /plate |
0 |
0 |
0 |
|
(LEMI code :20/0285-210920-S1) |
|||||
50 µg /plate |
0 |
0 |
0 |
||
without metabolic activation |
|||||
15 µg /plate |
0 |
0 |
0 |
||
Solution of |
5 µg /plate |
0 |
0 |
0 |
|
1.5 µg /plate |
0 |
0 |
0 |
||
T-A12 BATCH: I20196A |
0.5 µg /plate |
0 |
0 |
0 |
|
(LEMI code :20/0285-290920-S2) |
|||||
0.15 µg /plate |
0 |
0 |
0 |
||
with metabolic activation |
|||||
0.05 µg /plate |
0 |
0 |
0 |
||
S9-mix |
500 µL/plate |
0 |
0 |
0 |
|
Control n° 3 |
1 |
2 |
3 |
||
Solution of |
1500 µg /plate |
0 |
0 |
0 |
|
500 µg /plate |
0 |
0 |
0 |
||
T-A12 BATCH: I20196A |
150 µg /plate |
0 |
0 |
0 |
|
(LEMI code :20/0285-280920-S1) |
|||||
50 µg /plate |
0 |
0 |
0 |
||
without metabolic activation |
|||||
15 µg /plate |
0 |
0 |
0 |
||
Solution of |
5 µg /plate |
0 |
0 |
0 |
|
1.5 µg /plate |
0 |
0 |
0 |
||
T-A12 BATCH: I20196A |
0.5 µg /plate |
0 |
0 |
0 |
|
(LEMI code :20/0285-280920-S2) |
|||||
0.15 µg /plate |
0 |
0 |
0 |
||
with metabolic activation |
|||||
0.05 µg /plate |
0 |
0 |
0 |
||
S9-mix |
500 µL/plate |
0 |
0 |
0 |
Table 4.1. Bacteriostatic activity controls. TA100. Preliminary assay
|
Doses (/plate) |
||||||||||||||||
0 (negative control) |
DMSO |
50 µg |
150 µg |
500 µg |
1 500 µg |
2500 µg |
5 000 µg (p) |
||||||||||
|
N1 |
701 |
765 |
710 |
690 |
520 |
130 |
135 |
53 |
||||||||
Solution of |
N2 |
677 |
577 |
752 |
723 |
510 |
175 |
189 |
70 |
||||||||
T-A12 BATCH: I20196A |
N3 |
754 |
718 |
742 |
718 |
474 |
192 |
201 |
60 |
||||||||
|
N |
711±39 |
687±98 |
735±22 |
710±18 |
501±24 |
166±32 |
175±35 |
61±9 |
||||||||
LEMI code : 20/0285-030820-S1 |
% |
- |
|
97% |
|
103% |
100% |
|
71% |
|
23% |
|
25% |
|
|
9% |
|
|
Doses (/plate) |
|||||||||||||||
0 (negative control) |
DMSO |
500 µg |
1000 µg |
2000 µg |
3000 µg |
5 000 µg (p) |
||||||||||
|
N1 |
679 |
735 |
510 |
250 |
190 |
185 |
67 |
||||||||
Solution of |
N2 |
698 |
726 |
520 |
221 |
222 |
102 |
71 |
||||||||
T-A12 BATCH: I20196A |
N3 |
714 |
687 |
444 |
265 |
175 |
163 |
81 |
||||||||
|
N |
697±18 |
716±26 |
491±41 |
245±22 |
196±24 |
150±43 |
73±7 |
||||||||
LEMI code : 20/0285-170820-S1 |
% |
- |
103% |
|
70% |
|
|
35% |
|
|
28% |
|
22% |
|
10% |
|
(p) presence of precipitates
N1 Number of colonies in plate 1
N2 Number of colonies in plate 2
N3 Number of colonies in plate 3
N Mean per plate
% Percent of survival compared to negative control
Table 4.2. Bacteriostatic activity controls. Additional assays. Revertant colonies number
|
STRAINS without metabolic activation |
||||
Doses |
TA100 |
TA98 |
TA1537 |
TA1535 |
E. Coli |
5000µg/plate(p) |
0 |
0 |
0 |
5 |
2-8 |
3000 µg/plate |
3 |
3 |
1 |
0 |
2-4 |
1500 µg/plate |
1* |
6* |
0 |
2* |
32* |
500 µg/plate |
20 |
14 |
2 |
4 |
41 |
Spontaneous revertant colonies |
68-60 |
14 -23 |
2-7 |
18-8 |
43-46 |
|
STRAINS with metabolic activation |
||||
Doses |
TA100 |
TA98 |
TA1537 |
TA1535 |
E. Coli |
500 µg/plate |
0 |
0 |
0 |
0 |
0 |
250 µg/plate |
0 |
0 |
0 |
0 |
0 |
150 µg/plate |
0 |
0 |
0 |
0 |
0 |
50 µg/plate |
0 |
0 |
0 |
0 |
0 |
25 µg/plate |
0 |
7*** - 9*** |
1***-0*** |
0 |
11***-6*** |
10 µg/plate |
0 |
6**-3** |
x |
x |
6**-7** |
5 µg/plate |
3*-0* |
8*-1* |
1*-0* |
1*-0* |
15*-16* |
Spontaneous revertant colonies |
73 |
30 |
11 |
17 |
65 |
(p) presence of precipitates
*** high thinning of the bacterial lawn
** thinning of the bacterial lawn
*light thinning of the bacterial lawn
Table 4.3. Bacteriostatic activity controls. Additional assays. TA100
|
Doses (/plate) |
|||||||||||||||
0 (negative control) |
DMSO |
15 µg |
50 µg |
150 µg |
500 µg |
1500 µg |
||||||||||
|
N1 |
760 |
717 |
705 |
734 |
761 |
526 |
198 |
||||||||
Solution of |
N2 |
699 |
701 |
711 |
731 |
697 |
544 |
200 |
||||||||
T-A12 BATCH: I20196A |
N3 |
687 |
703 |
689 |
682 |
599 |
510 |
166 |
||||||||
|
N |
715±39 |
707±9 |
702±11 |
716±29 |
686±82 |
527±17 |
188±19 |
||||||||
LEMI code : 20/0285-210920-S1 |
% |
- |
|
99% |
|
|
98% |
|
100% |
|
96% |
|
74% |
|
26% |
|
|
Doses (/plate) |
|||||||||||||||||
0 (negative control) |
DMSO |
15 µg |
50 µg |
150 µg |
500 µg |
1500 µg |
||||||||||||
|
N1 |
761 |
687 |
638 |
644 |
588 |
475 |
162 |
||||||||||
Solution of |
N2 |
774 |
716 |
574 |
655 |
584 |
423 |
174 |
||||||||||
T-A12 BATCH: I20196A |
N3 |
714 |
728 |
686 |
618 |
593 |
463 |
178 |
||||||||||
|
N |
750±32 |
710±21 |
633±56 |
639±19 |
588±5 |
454±27 |
171±8 |
||||||||||
LEMI code : 20/0285-280920-S1 |
% |
- |
|
95% |
|
|
84% |
|
|
85% |
|
|
78% |
|
61% |
|
23% |
|
Table 5. Result tables.
TA1535 Assay n°2 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
5 |
16 |
9 |
10.00 |
5.57 |
_ |
Positive control solvent |
5 µL |
13 |
11 |
11 |
11.67 |
1.15 |
_ |
Positive control: Sodium azide |
5 µg in 5 µL |
653 |
699 |
670 |
674.00 |
23.26 |
57.77 |
Vehicle |
50 µL |
8 |
5 |
5 |
6.00 |
1.73 |
_ |
|
1500 µg* |
1 |
5 |
1 |
2.33 |
2.31 |
0.39 |
Solution of |
500 µg* |
7 |
5 |
3 |
5.00 |
2.00 |
0.83 |
T-A12 BATCH: I20196A |
150 µg |
10 |
15 |
9 |
11.33 |
3.21 |
1.89 |
|
50 µg |
8 |
5 |
12 |
8.33 |
3.51 |
1.39 |
LEMI code :20/0285-210920-S1 |
15 µg |
9 |
9 |
4 |
7.33 |
2.89 |
1.22 |
TA1535 Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
13 |
11 |
13 |
12.33 |
1.15 |
_ |
Positive control solvent |
20 µL |
8 |
12 |
13 |
11.00 |
2.65 |
_ |
Positive control: 2-Anthramine |
2 µg in 20 µL |
197 |
194 |
188 |
193.00 |
4.58 |
17.55 |
Vehicle |
50 µL |
7 |
4 |
15 |
8.67 |
5.69 |
_ |
|
5 µg** |
4 |
3 |
4 |
3.67 |
0.58 |
0.42 |
Solution of |
1.5 µg* |
5 |
6 |
8 |
6.33 |
1.53 |
0.73 |
T-A12 BATCH: I20196A |
0.5 µg |
9 |
7 |
12 |
9.33 |
2.52 |
1.08 |
|
0.15µg |
13 |
9 |
5 |
9.00 |
4.00 |
1.04 |
LEMI code :20/0285-210920-S2 |
0.05 µg |
11 |
10 |
13 |
11.33 |
1.53 |
1.31 |
TA1535 Assay n°3 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
6 |
5 |
10 |
7.00 |
2.65 |
_ |
Positive control solvent |
5 µL |
5 |
11 |
11 |
9.00 |
3.46 |
_ |
Positive control: Sodium azide |
5 µg in 5 µL |
834 |
793 |
810 |
812.33 |
20.60 |
90.26 |
Vehicle |
50 µL |
8 |
9 |
5 |
7.33 |
2.08 |
_ |
|
1500 µg* |
3 |
2 |
1 |
2.00 |
1.00 |
0.27 |
Solution of |
500 µg* |
8 |
6 |
3 |
5.67 |
2.52 |
0.77 |
T-A12 BATCH: I20196A |
150 µg |
4 |
14 |
7 |
8.33 |
5.13 |
1.14 |
|
50 µg |
11 |
9 |
8 |
9.33 |
1.53 |
1.27 |
LEMI code :20/0285-280920-S1 |
15 µg |
8 |
9 |
7 |
8.00 |
1.00 |
1.09 |
TA1535 Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
14 |
7 |
7 |
9.33 |
4.04 |
_ |
Positive control solvent |
10 µL |
12 |
7 |
8 |
9.00 |
2.65 |
_ |
Positive control: 2-Anthramine |
1 µg in 10 µL |
64 |
48 |
40 |
50.67 |
12.22 |
5.63 |
Vehicle |
50 µL |
9 |
7 |
9 |
8.33 |
1.15 |
_ |
|
5 µg*** |
1 |
2 |
1 |
1.33 |
0.58 |
0.16 |
Solution of |
1.5 µg** |
4 |
5 |
1 |
3.33 |
2.08 |
0.40 |
T-A12 BATCH: I20196A |
0.5 µg* |
10 |
9 |
8 |
9.00 |
1.00 |
1.08 |
|
0.15µg |
9 |
4 |
5 |
6.00 |
2.65 |
0.72 |
LEMI code :20/0285-280920-S2 |
0.05 µg |
12 |
7 |
9 |
9.33 |
2.52 |
1.12 |
TA1537 Assay n°2 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
11 |
12 |
7 |
10.00 |
2.65 |
_ |
Positive control solvent |
20 µL |
14 |
12 |
13 |
13.00 |
1.00 |
_ |
Positivecontrol: 9-Aminoacridine |
50 µg in 20 µL |
1569 |
904 |
1495 |
1322.67 |
364.46 |
101.74 |
Vehicle |
50 µL |
11 |
7 |
8 |
8.67 |
2.08 |
_ |
|
1500 µg* |
2 |
1 |
2 |
1.67 |
0.58 |
0.19 |
Solution of |
500 µg |
5 |
8 |
5 |
6.00 |
1.73 |
0.69 |
T-A12 BATCH: I20196A |
150 µg |
9 |
7 |
6 |
7.33 |
1.53 |
0.85 |
|
50 µg |
8 |
7 |
12 |
9.00 |
2.65 |
1.04 |
LEMI code :20/0285-210920-S1 |
15 µg |
10 |
9 |
12 |
10.33 |
1.53 |
1.19 |
TA1537 Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
8 |
6 |
18 |
10.67 |
6.43 |
_ |
Positive control solvent |
20 µL |
15 |
8 |
7 |
10.00 |
4.36 |
_ |
Positive control: 2-Anthramine |
2 µg in 20 µL |
73 |
71 |
60 |
68.00 |
7.00 |
6.80 |
Vehicle |
50 µL |
13 |
14 |
15 |
14.00 |
1.00 |
_ |
|
5 µg** |
3 |
4 |
4 |
3.67 |
0.58 |
0.26 |
Solution of |
1.5 µg* |
11 |
3 |
4 |
6.00 |
4.36 |
0.43 |
T-A12 BATCH: I20196A |
0.5 µg |
6 |
9 |
8 |
7.67 |
1.53 |
0.55 |
|
0.15µg |
8 |
13 |
11 |
10.67 |
2.52 |
0.76 |
LEMI code :20/0285-210920-S2 |
0.05 µg |
7 |
9 |
11 |
9.00 |
2.00 |
0.64 |
TA1537 Assay n°3 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
5 |
9 |
6 |
6.67 |
2.08 |
_ |
Positive control solvent |
20 µL |
2 |
9 |
6 |
5.67 |
3.51 |
_ |
Positivecontrol: 9-Aminoacridine |
50 µg in 20 µL |
918 |
1461 |
834 |
1071.00 |
340.35 |
189.00 |
Vehicle |
50 µL |
9 |
2 |
4 |
5.00 |
3.61 |
_ |
|
1500 µg* |
2 |
1 |
1 |
1.33 |
0.58 |
0.27 |
Solution of |
500 µg |
5 |
2 |
3 |
3.33 |
1.53 |
0.67 |
T-A12 BATCH: I20196A |
150 µg |
3 |
2 |
2 |
2.33 |
0.58 |
0.47 |
|
50 µg |
5 |
4 |
8 |
5.67 |
2.08 |
1.13 |
LEMI code :20/0285-280920-S1 |
15 µg |
7 |
5 |
7 |
6.33 |
1.15 |
1.27 |
TA1537 Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
7 |
12 |
9 |
9.33 |
2.52 |
_ |
Positive control solvent |
10 µL |
4 |
4 |
3 |
3.67 |
0.58 |
_ |
Positive control : 2-Anthramine |
1 µg in 10 µL |
62 |
59 |
51 |
57.33 |
5.69 |
15.64 |
Vehicle |
50 µL |
4 |
3 |
7 |
4.67 |
2.08 |
_ |
|
5 µg*** |
4 |
2 |
4 |
3.33 |
1.15 |
0.71 |
Solution of |
1.5 µg** |
6 |
2 |
2 |
3.33 |
2.31 |
0.71 |
T-A12 BATCH: I20196A |
0.5 µg* |
7 |
4 |
9 |
6.67 |
2.52 |
1.43 |
|
0.15 µg |
4 |
5 |
7 |
5.33 |
1.53 |
1.14 |
LEMI code :20/0285-280920-S2 |
0.05 µg |
5 |
4 |
3 |
4.00 |
1.00 |
0.86 |
TA98 Assay n°2 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
17 |
20 |
20 |
19.00 |
1.73 |
_ |
Positive control solvent |
20 µL |
24 |
22 |
26 |
24.00 |
2.00 |
_ |
Positivecontrol: 2-Nitrofluorene |
2 µg in 20 µL |
258 |
381 |
281 |
306.67 |
65.39 |
12.78 |
Vehicle |
50 µL |
19 |
20 |
23 |
20.67 |
2.08 |
_ |
|
1500 µg* |
9 |
13 |
16 |
12.67 |
3.51 |
0.61 |
Solution of |
500 µg |
14 |
16 |
22 |
17.33 |
4.16 |
0.84 |
T-A12 BATCH: I20196A |
150 µg |
18 |
15 |
18 |
17.00 |
1.73 |
0.82 |
|
50 µg |
21 |
24 |
16 |
20.33 |
4.04 |
0.98 |
LEMI code :20/0285-210920-S1 |
15 µg |
22 |
20 |
20 |
20.67 |
1.15 |
1.00 |
TA98 Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
27 |
35 |
38 |
33.33 |
5.69 |
_ |
Positive control solvent |
20 µL |
35 |
31 |
37 |
34.33 |
3.06 |
_ |
Positive control : 2-Anthramine |
2 µg in 20 µL |
566 |
526 |
704 |
598.67 |
93.39 |
17.44 |
Vehicle |
50 µL |
37 |
31 |
36 |
34.67 |
3.21 |
_ |
|
5 µg* |
7 |
8 |
3 |
6.00 |
2.65 |
0.17 |
Solution of |
1,5 µg |
10 |
18 |
13 |
13.67 |
4.04 |
0.39 |
T-A12 BATCH: I20196A |
0.5 µg |
30 |
28 |
40 |
32.67 |
6.43 |
0.94 |
|
0,15 µg |
33 |
41 |
34 |
36.00 |
4.36 |
1.04 |
LEMI code :20/0285-210920-S2 |
0.05 µg |
47 |
28 |
29 |
34.67 |
10.69 |
1.00 |
TA98 Assay n°3 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
6 |
8 |
7 |
7.00 |
1.00 |
_ |
Positive control solvent |
20 µL |
9 |
6 |
9 |
8.00 |
1.73 |
_ |
Positivecontrol: 2-Nitrofluorene |
2 µg in 20 µL |
254 |
252 |
315 |
273.67 |
35.81 |
34.21 |
Vehicle |
50 µL |
9 |
9 |
6 |
8.00 |
1.73 |
_ |
|
1500 µg* |
2 |
14 |
7 |
7.67 |
6.03 |
0.96 |
Solution of |
500 µg |
5 |
8 |
11 |
8.00 |
3.00 |
1.00 |
T-A12 BATCH: I20196A |
150 µg |
10 |
11 |
9 |
10.00 |
1.00 |
1.25 |
|
50 µg |
8 |
10 |
10 |
9.33 |
1.15 |
1.17 |
LEMI code :20/0285-280920-S1 |
15 µg |
10 |
11 |
8 |
9.67 |
1.53 |
1.21 |
TA98 Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
14 |
10 |
15 |
13.00 |
2.65 |
_ |
Positive control solvent |
10 µL |
10 |
12 |
10 |
10.67 |
1.15 |
_ |
Positive control: 2-Anthramine |
1 µg in 10 µL |
255 |
257 |
219 |
243.67 |
21.39 |
22.84 |
Vehicle |
50 µL |
14 |
14 |
11 |
13.00 |
1.73 |
_ |
|
5 µg** |
14 |
18 |
6 |
12.67 |
6.11 |
0.97 |
Solution of |
1,5 µg* |
24 |
16 |
4 |
14.67 |
10.07 |
1.13 |
T-A12 BATCH: I20196A |
0,5 µg |
19 |
14 |
11 |
14.67 |
4.04 |
1.13 |
|
0,15 µg |
14 |
19 |
13 |
15.33 |
3.21 |
1.18 |
LEMI code :20/0285-280920-S2 |
0.05 µg |
12 |
12 |
12 |
12.00 |
0.00 |
0.92 |
TA100 Assay n°2 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
66 |
71 |
65 |
67.33 |
3.21 |
_ |
Positive control solvent |
20 µL |
70 |
59 |
60 |
63.00 |
6.08 |
_ |
Positive control: Sodium azide |
20 µg in 20 µL |
1246 |
1125 |
1233 |
1201.33 |
66.43 |
19.07 |
Vehicle |
50 µL |
65 |
61 |
62 |
62.67 |
2.08 |
_ |
|
1500 µg** |
6 |
2 |
4 |
4.00 |
2.00 |
0.06 |
Solution of |
500 µg* |
18 |
27 |
18 |
21.00 |
5.20 |
0.34 |
T-A12 BATCH: I20196A |
150 µg* |
67 |
52 |
70 |
63.00 |
9.64 |
1.01 |
|
50 µg |
58 |
71 |
67 |
65.33 |
6.66 |
1.04 |
LEMI code :20/0285-210920-S1 |
15 µg |
67 |
59 |
56 |
60.67 |
5.69 |
0.97 |
TA100 Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
85 |
70 |
72 |
75.67 |
8.14 |
_ |
Positive control solvent |
20 µL |
70 |
75 |
69 |
71.33 |
3.21 |
_ |
Positive control: 2-Anthramine |
2 µg in 20 µL |
629 |
1051 |
954 |
878.00 |
221.03 |
12.31 |
Vehicle |
50 µL |
80 |
77 |
73 |
76.67 |
3.51 |
_ |
|
5 µg** |
4 |
6 |
7 |
5.67 |
1.53 |
0.07 |
Solution of |
1.5 µg** |
17 |
12 |
12 |
13.67 |
2.89 |
0.18 |
T-A12 BATCH: I20196A |
0.5 µg* |
70 |
79 |
90 |
79.67 |
10.02 |
1.04 |
|
0.15 µg |
83 |
83 |
66 |
77.33 |
9.81 |
1.01 |
LEMI code :20/0285-210920-S2 |
0.05 µg |
70 |
64 |
62 |
65.33 |
4.16 |
0.85 |
TA100 Assay n°3 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
52 |
70 |
59 |
60.33 |
9.07 |
_ |
Positive control solvent |
20 µL |
56 |
62 |
62 |
60.00 |
3.46 |
_ |
Positive control: Sodium azide |
20 µg in 20 µL |
1029 |
1284 |
1024 |
1112.33 |
148.69 |
18.54 |
Vehicle |
50 µL |
46 |
51 |
48 |
48.33 |
2.52 |
_ |
|
1500 µg** |
2 |
5 |
6 |
4.33 |
2.08 |
0.09 |
Solution of |
500 µg* |
8 |
12 |
15 |
11.67 |
3.51 |
0.24 |
T-A12 BATCH: I20196A |
150 µg* |
79 |
56 |
36 |
57.00 |
21.52 |
1.18 |
|
50 µg |
56 |
75 |
65 |
65.33 |
9.50 |
1.35 |
LEMI code :20/0285-280920-S1 |
15 µg |
80 |
69 |
72 |
73.67 |
5.69 |
1.52 |
TA100 Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
72 |
71 |
63 |
68.67 |
4.93 |
_ |
Positive control solvent |
10 µL |
86 |
88 |
73 |
82.33 |
8.14 |
_ |
Positive control: 2-Anthramine |
1 µg in 10 µL |
297 |
370 |
301 |
322.67 |
41.04 |
3.92 |
Vehicle |
50 µL |
72 |
63 |
84 |
73.00 |
10.54 |
_ |
|
5 µg*** |
4 |
6 |
5 |
5.00 |
1.00 |
0.07 |
Solution of |
1.5 µg** |
10 |
18 |
13 |
13.67 |
4.04 |
0.19 |
T-A12 BATCH: I20196A |
0.5 µg* |
55 |
53 |
49 |
52.33 |
3.06 |
0.72 |
|
0.15 µg |
79 |
79 |
74 |
77.33 |
2.89 |
1.06 |
LEMI code :20/0285-280920-S2 |
0.05 µg |
66 |
86 |
69 |
73.67 |
10.79 |
1.01 |
E. COLI Assay n°2 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
68 |
115 |
95 |
92.67 |
23.59 |
_ |
Positive control solvent |
10 µL |
84 |
100 |
104 |
96.00 |
10.58 |
_ |
Positivecontrol: cis-Platinum(II) |
1 µg in 10 µL |
409 |
511 |
399 |
439.67 |
61.98 |
4.58 |
Vehicle |
50 µL |
112 |
87 |
87 |
95.33 |
14.43 |
_ |
|
1500 µg* |
26 |
25 |
17 |
22.67 |
4.93 |
0.24 |
Solution of |
500 µg |
70 |
75 |
51 |
65.33 |
12.66 |
0.69 |
T-A12 BATCH: I20196A |
150 µg |
76 |
81 |
71 |
76.00 |
5.00 |
0.80 |
|
50 µg |
93 |
85 |
84 |
87.33 |
4.93 |
0.92 |
LEMI code :20/0285-210920-S1 |
15 µg |
62 |
70 |
75 |
69.00 |
6.56 |
0.72 |
E. COLI Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
68 |
96 |
95 |
86.33 |
15.89 |
_ |
Positive control solvent |
5 µL |
94 |
98 |
98 |
96.67 |
2.31 |
_ |
Positive control: Dimethylbenzanthracene |
5 µg in 5 µL |
608 |
599 |
566 |
591.00 |
22.11 |
6.11 |
Vehicle |
50 µL |
93 |
115 |
84 |
97.33 |
15.95 |
_ |
|
5 µg* |
29 |
46 |
27 |
34.00 |
10.44 |
0.35 |
Solution of |
1,5 µg |
75 |
91 |
53 |
73.00 |
19.08 |
0.75 |
T-A12 BATCH: I20196A |
0.5 µg |
100 |
102 |
110 |
104.00 |
5.29 |
1.07 |
|
0.15 µg |
97 |
98 |
105 |
100.00 |
4.36 |
1.03 |
LEMI code :20/0285-210920-S2 |
0.05 µg |
97 |
105 |
73 |
91.67 |
16.65 |
0.94 |
E. COLI Assay n°3 – without metabolic activation (-S9-mix) |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
60 |
56 |
53 |
56.33 |
3.51 |
_ |
Positive control solvent |
10 µL |
59 |
58 |
54 |
57.00 |
2.65 |
_ |
Positivecontrol: cis-Platinum(II) |
1 µg in 10 µL |
424 |
389 |
412 |
408.33 |
17.79 |
7.16 |
Vehicle |
50 µL |
44 |
59 |
68 |
57.00 |
12.12 |
_ |
|
1500 µg* |
30 |
23 |
29 |
27.33 |
3.79 |
0.48 |
Solution of |
500 µg |
57 |
63 |
51 |
57.00 |
6.00 |
1.00 |
T-A12 BATCH: I20196A |
150 µg |
58 |
61 |
71 |
63.33 |
6.81 |
1.11 |
|
50 µg |
47 |
55 |
57 |
53.00 |
5.29 |
0.93 |
LEMI code :20/0285-280920-S1 |
15 µg |
45 |
41 |
42 |
42.67 |
2.08 |
0.75 |
E. COLI Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation |
|||||||
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
n° 1 |
n° 2 |
n° 3 |
|||||
Negative control |
100 µL |
69 |
74 |
91 |
78.00 |
11.53 |
_ |
Positive control solvent |
5 µL |
77 |
77 |
70 |
74.67 |
4.04 |
_ |
Positive control : Dimethylbenzanthracene |
2.5 µg in 5 µL |
389 |
415 |
437 |
413.67 |
24.03 |
5.54 |
Vehicle |
50 µL |
75 |
79 |
69 |
74.33 |
5.03 |
_ |
|
5 µg** |
21 |
23 |
20 |
21.33 |
1.53 |
0.29 |
Solution of |
1,5 µg* |
73 |
79 |
89 |
80.33 |
8.08 |
1.08 |
T-A12 BATCH: I20196A |
0,5 µg |
88 |
77 |
114 |
93.00 |
19.00 |
1.25 |
|
0.15 µg |
81 |
70 |
74 |
75.00 |
5.57 |
1.01 |
LEMI code :20/0285-280920-S2 |
0.05 µg |
75 |
69 |
88 |
77.33 |
9.71 |
1.04 |
*** high thinning of the bacterial lawn
** thinning of the bacterial lawn
*light thinning of the bacterial lawn
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Based on the available data (negative Ames test), the test
substance is not classified for mutagenecity in accordance with CLP
Regulation (EC) No. 1272/2008.
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