Bentonite, acid-leached

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:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

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

Test material information

Constituent 1

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

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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.

Controls

Controls 1

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

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 80 μg/plate for TA1537 - without S9-mix

Controls 3

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 0.2 μg/plate for TA98 - without S9-mix

Controls 4

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-

Controls 5

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 results

Test results 1

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

Test results 2

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 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	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 deviation

Applicant'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.