Registration Dossier
Registration Dossier
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 274-324-8 | CAS number: 70131-50-9
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:
- The experimental phase of the study was performed between 11 February 2010 and 25 March 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study performed according to GLP and internationally accepted guideline
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
- Report date:
- 2010
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Bentonite, acid-leached
- EC Number:
- 274-324-8
- EC Name:
- Bentonite, acid-leached
- Cas Number:
- 70131-50-9
- Molecular formula:
- unspecified for this UVCB substance
- IUPAC Name:
- Bentonite, acid-leached
- Details on test material:
- - Name of test material (as cited in study report):Bentonite acid-leached
- Physical state: Solid
- Purity.: 100%
- Expiration date of the lot/batch: 31 December 2010
- Storage condition of test material: Room temperature in the dark
Constituent 1
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (rat liver homogenate metabolising system (10% liver S9 in standard co-factors))
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Mutation test - Experiments 1 and 2: 50, 150, 500, 1500 and 5000 μg/plate - Vehicle / solvent:
- - Vehicle: Polyethylene glycol 400
- Justification for choice of vehicle: For compounds that are completely insoluble in the standard Ames solvents as recommended by Maron et al (1980) the testing laboratory found that Polyethylene glycol 400 is an excellent suspending agent. However, as this vehicle is not on the recommended lists for acceptability, in-house testing was performed to ensure that PEG 400 is non-toxic to either the bacteria or the microsomal S9 enzymes and that it does not inhibit the mutagenic effect of the standard positive controls. All results were within acceptable ranges and therefore PEG 400 is considered to be satisfactory for use in the reverse mutation assay.
- Justification for choice of vehicle: The test material was insoluble and could not be sufficiently suspended in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in-house. The test material formed the best doseable suspension in polyethylene glycol 400 and was therefore selected as the vehicle.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- Concurrent untreated controls
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Migrated to IUCLID6: 2 μg/plate for WP2uvrA-, 3 μg/plate for TA100, 5 μg/plate for TA1535 - without S9-mix
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Migrated to IUCLID6: 80 μg/plate for TA1537 - without S9-mix
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- Migrated to IUCLID6: 0.2 μg/plate for TA98 - without S9-mix
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA) with S9-mix: 1 μg/plate for TA100; 2 μg/plate for TA1535 and TA1537; 10 μg/plate for WP2uvrA-
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- Migrated to IUCLID6: 5 μg/plate for TA98 - with S9-mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and preincubation for Experiment 2:
Preliminary Toxicity Test: In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA-), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
Mutation Test - Experiment 1: Five concentrations of the test material (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Mutation Test - Experiment 2: The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 μg/plate).
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and foreach concentration of test material both with and without S9-mix. Manual counts were required after employing the pre-incubation method at 5000 μg/plate (absence of S9-mix only) because of a particulate test material precipitation.
DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48 h (Experiment 1)
NUMBER OF REPLICATIONS: Plates were prepared in triplicate
NUMBER OF CELLS EVALUATED: All tester strain cultures should be in the range of 1 to 9.9 x 10^09 bacteria per mL. - Evaluation criteria:
- There were several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results were considered first, statistical methods, as recommended by the UKEMS were also used as an aid to evaluation, however, statistical significance was not the only determining factor for a positive response.
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
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The vehicle (polyethylene glycol 400) 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 test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A particulate precipitate was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9 at 50 μg/plate in Experiment 2. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.19 times the concurrent vehicle control. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table1: Spontaneous Mutation Rates (Concurrent Negative Controls)
EXPERIMENT 1
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||
97 |
|
20 |
|
22 |
|
18 |
|
7 |
|
91 |
(96) |
24 |
(21) |
29 |
(24) |
23 |
(19) |
11 |
(11) |
99 |
|
19 |
|
20 |
|
15 |
|
14 |
|
EXPERIMENT 2
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||
114 |
|
21 |
|
25 |
|
15 |
|
14 |
|
105 |
(102) |
24 |
(22) |
13 |
(19) |
12 |
(17) |
12 |
(12) |
86 |
|
20 |
|
19 |
|
25 |
|
11 |
|
Table2 Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 15 March 2010 |
To: 18 March 2010 |
|||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||||
Base-pair substitution type |
Frameshift type |
||||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||||
- |
0 |
129 130 115 |
(125) 8.4# |
20 25 22 |
(22) 2.5 |
23 33 29 |
(28) 5.0 |
26 21 35 |
(27) 7.1 |
10 11 14 |
(12) 2.1 |
||
- |
50 |
124 109 113 |
(115) 7.8 |
24 25 19 |
(23) 3.2 |
26 14 20 |
(20) 6.0 |
21 20 22 |
(21) 1.0 |
11 10 11 |
(11) 0.6 |
||
- |
150 |
115 111 119 |
(115) 4.0 |
27 23 30 |
(27) 3.5 |
23 25 20 |
(23) 2.5 |
24 23 33 |
(27) 5.5 |
14 10 10 |
(11) 2.3 |
||
- |
500 |
135 101 119 |
(118) 17.0 |
19 29 30 |
(26) 6.1 |
24 33 31 |
(29) 4.7 |
21 26 23 |
(23) 2.5 |
16 11 15 |
(14) 2.6 |
||
- |
1500 |
111 P 137 P 119 P |
(122) 13.3 |
36 P 23 P 13 P |
(24) 11.5 |
25 P 16 P 22 P |
(21) 4.6 |
30 P 26 P 24 P |
(27) 3.1 |
11 P 8 P 12 P |
(10) 2.1 |
||
- |
5000 |
108 P 126 P 111 P |
(115) 9.6 |
20 P 23 P 25 P |
(23) 2.5 |
25 P 25 P 28 P |
(26) 1.7 |
23 P 21 P 24 P |
(23) 1.5 |
7 P 12 P 13 P |
(11) 3.2 |
||
Positive controls
S9-Mix
- |
Name Concentration (μg/plate) No. colonies per plate |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||||
3 |
5 |
2 |
0.2 |
80 |
|||||||||
586 624 731 |
(647) 75.2 |
154 111 162 |
(142) 27.4 |
242 227 242 |
(237) 8.7 |
102 95 118 |
(105) 11.8 |
1270 1027 935 |
(1077) 173.1 |
||||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Precipitate
# Standard deviation Table 3: Test results: Experiment 1 - With metabolic activationTest Period |
From: 15 March 2010 |
To: 18 March 2010 |
||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
||||||||
+ |
0 |
106 130 119 |
(118) 12.0# |
24 15 12 |
(17) 6.2 |
23 21 30 |
(25) 4.7 |
29 26 27 |
(27) 1.5 |
14 12 16 |
(14) 2.0 |
|
+ |
50 |
117 108 112 |
(112) 4.5 |
14 25 15 |
(18) 6.1 |
20 23 22 |
(22) 1.5 |
26 33 23 |
(27) 5.1 |
18 12 14 |
(15) 3.1 |
|
+ |
150 |
103 114 101 |
(106) 7.0 |
13 12 19 |
(15) 3.8 |
22 16 22 |
(20) 3.5 |
35 20 24 |
(26) 7.8 |
8 8 11 |
(9) 1.7 |
|
+ |
500 |
98 104 113 |
(105) 7.5 |
18 19 21 |
(19) 1.5 |
21 33 24 |
(26) 6.2 |
30 31 27 |
(29) 2.1 |
15 11 9 |
(12) 3.1 |
|
+ |
1500 |
128 P 97 P 135 P |
(120) 20.2 |
18 P 16 P 23 P |
(19) 3.6 |
26 P 33 P 24 P |
(28) 4.7 |
31 P 27 P 29 P |
(29) 2.0 |
13 P 15 P 11 P |
(13) 2.0 |
|
+ |
5000 |
117 P 121 P 123 P |
(120) 3.1 |
19 P 16 P 15 P |
(17) 2.1 |
22 P 28 P 26 P |
(25) 3.1 |
25 P 21 P 23 P |
(23) 2.0 |
12 P 11 P 9 P |
(11) 1.5 |
|
Positive controls
S9-Mix
+ |
Name Concentration (μg/plate) No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 |
2 |
10 |
5 |
2 |
||||||||
305 310 280 |
(298) 16.1 |
145 200 156 |
(167) 29.1 |
181 146 184 |
(170) 21.1 |
168 148 119 |
(145) 24.6 |
131 124 155 |
(137) 16.3 |
|||
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
P Precipitate
# Standard deviation
Table 4: Test results: Experiment 2 - Without metabolic activation
Test Period |
From: 22 March 2010 |
To: 25 March 2010 |
|||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||||
Base-pair substitution type |
Frameshift type |
||||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||||
- |
0 |
142 126 128 |
(132) 8.7# |
13 21 13 |
(16) 4.6 |
20 12 26 |
(19) 7.0 |
24 16 22 |
(21) 4.2 |
5 10 8 |
(8) 2.5 |
||
- |
50 |
130 124 106 |
(120) 12.5 |
24 23 24 |
(24) 0.6 |
14 33 15 |
(21) 10.7 |
29 19 24 |
(24) 5.0 |
12 5 4 |
(7) 4.4 |
||
- |
150 |
135 129 110 |
(125) 13.1 |
13 22 14 |
(16) 4.9 |
18 12 19 |
(16) 3.8 |
20 34 25 |
(26) 7.1 |
14 12 4 |
(10) 5.3 |
||
- |
500 |
156 128 154 |
(146) 15.6 |
23 12 15 |
(17) 5.7 |
27 22 26 |
(25) 2.6 |
27 14 25 |
(22) 7.0 |
3 1 7 |
(4) 3.1 |
||
- |
1500 |
148 P 139 P 135 P |
(141) 6.7 |
21 P 15 P 14 P |
(17) 3.8 |
22 P 22 P 27 P |
(24) 2.9 |
32 P 29 P 32 P |
(31) 1.7 |
7 P 15 P 8 P |
(10) 4.4 |
||
- |
5000 |
135 P 124 P 150 P |
(136) 13.1 |
18 P 12 P 18 P |
(16) 3.5 |
18 P 29 P 23 P |
(23) 5.5 |
20 P 24 P 29 P |
(24) 4.5 |
3 P 12 P 9 P |
(8) 4.6 |
||
Positive controls
S9-Mix
- |
Name Concentration (μg/plate) No. colonies per plate |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||||
3 |
5 |
2 |
0.2 |
80 |
|||||||||
438 422 470 |
(443) 24.4 |
366 385 443 |
(398) 40.1 |
605 584 623 |
(604) 19.5 |
178 196 184 |
(186) 9.2 |
2484 3236 2411 |
(2710) 456.7 |
||||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Precipitate
# Standard deviation
Table 5: Test results: Experiment 2 - With metabolic activation
Test Period |
From: 22 March 2010 |
To: 25 March 2010 |
||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
||||||||
+ |
0 |
153 168 139 |
(153) 14.5# |
18 11 11 |
(13) 4.0 |
27 24 25 |
(25) 1.5 |
35 32 30 |
(32) 2.5 |
12 16 9 |
(12) 3.5 |
|
+ |
50 |
168 200 178 |
* (182) 16.4 |
21 14 13 |
(16) 4.4 |
22 19 16 |
(19) 3.0 |
30 27 20 |
(26) 5.1 |
14 16 9 |
(13) 3.6 |
|
+ |
150 |
141 136 132 |
(136) 4.5 |
10 13 20 |
(14) 5.1 |
19 23 20 |
(21) 2.1 |
25 42 26 |
(31) 9.5 |
9 22 4 |
(12) 9.3 |
|
+ |
500 |
144 153 162 |
(153) 9.0 |
16 22 21 |
(20) 3.2 |
32 34 30 |
(32) 2.0 |
30 33 36 |
(33) 3.0 |
9 15 8 |
(11) 3.8 |
|
+ |
1500 |
139 P 113 P 135 P |
(129) 14.0 |
24 P 11 P 20 P |
(18) 6.7 |
32 P 37 P 16 P |
(28) 11.0 |
43 P 23 P 48 P |
(38) 13.2 |
12 P 7 P 10 P |
(10) 2.5 |
|
+ |
5000 |
139 P 140 P 159 P |
(146) 11.3 |
12 P 14 P 14 P |
(13) 1.2 |
19 P 18 P 16 P |
(18) 1.5 |
38 P 21 P 23 P |
(27) 9.3 |
11 P 3 P 8 P |
(7) 4.0 |
|
Positive controls
S9-Mix
+ |
Name Concentration (μg/plate) No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 |
2 |
10 |
5 |
2 |
||||||||
1399 1269 1343 |
(1337) 65.2 |
249 202 265 |
(239) 32.8 |
297 283 284 |
(288) 7.8 |
379 321 323 |
(341) 32.9 |
319 227 180 |
(242) 70.7 |
|||
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
P Precipitate
* p£0.05
# Standard deviationApplicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
The test material was considered to be non-mutagenic under the conditions of this test.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
