Kieselguhr, soda ash flux-calcined

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:

Study was performed between 25 February 2010 and 23 April 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:

Kieselguhr, soda ash flux-calcined

EC Number:

272-489-0

EC Name:

Kieselguhr, soda ash flux-calcined

Cas Number:

68855-54-9

Molecular formula:

SiO2

IUPAC Name:

silanedione

Details on test material:

Sponsor's identification: Soda-ash flux calcined kieselguhr
Description : White powder
Purity : 100%
Batch number : 211184
Date received : 10 February 2010
Expiry date : 31 December 2013
Storage conditions: 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.

The test material was insoluble 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 a good, doseable suspension in polyethylene glycol 400 at 12.5 and 25 mg/ml, therefore, this suspending agent was 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 are 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 will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be 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 test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

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 fine, black particulate precipitate was noted at 5000 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 strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

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
93		23		29		23		8
88	(91)	24	(23)	30	(27)	18	(20)	11	(10)
93		22		21		20		12

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
125		27		29		23		15
120	(114)	22	(24)	31	(26)	21	(22)	8	(12)
98		22		19		23		13

 

Table2:   Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 12 March 2010					To: 15 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	144 167 175	(162) 16.1#	22 27 18	(22) 4.5	29 20 21		(23) 4.9	32 22 34	(29) 6.4	23 16 16	(18) 4.0
-	50	187 174 181	(181) 6.5	23 18 20	(20) 2.5	21 29 20		(23) 4.9	26 29 33	(29) 3.5	20 20 12	(17) 4.6
-	150	161 181 152	(165) 14.8	21 18 23	(21) 2.5	21 21 19		(20) 1.2	22 26 32	(27) 5.0	18 16 15	(16) 1.5
-	500	191 173 141	(168) 25.3	22 21 23	(22) 1.0	18 26 16		(20) 5.3	27 22 29	(26) 3.6	15 19 20	(18) 2.6
-	1500	166 166 153	(162) 7.5	16 24 23	(21) 4.4	26 27 26		(26) 0.6	29 16 27	(24) 7.0	19 16 21	(19) 2.5
-	5000	148 P 139 P 137 P	(141) 5.9	20 P 16 P 21 P	(19) 2.6	25 P 19 P 24 P		(23) 3.2	23 P 32 P 21 P	(25) 5.9	18 P 16 P 18 P	(17) 1.2
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		528 665 583	(592) 68.9	301 305 283	(296) 11.7	197 188 187		(191) 5.5	128 118 119	(122) 5.5	618 673 649	(647) 27.6

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

#        Standard deviation Table3              Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 12 March 2010					To: 15 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	185 157 172	(171) 14.0#	13 13 14	(13) 0.6	27 29 26		(27) 1.5	33 26 34	(31) 4.4	15 10 11	(12) 2.6
+	50	168 168 176	(171) 4.6	18 14 12	(15) 3.1	21 21 18		(20) 1.7	27 30 27	(28) 1.7	13 11 12	(12) 1.0
+	150	174 147 185	(169) 19.6	10 13 11	(11) 1.5	18 22 24		(21) 3.1	31 32 33	(32) 1.0	18 13 12	(14) 3.2
+	500	179 156 174	(170) 12.1	14 11 15	(13) 2.1	16 26 24		(22) 5.3	30 23 31	(28) 4.4	14 13 13	(13) 0.6
+	1500	185 180 136	(167) 27.0	13 16 13	(14) 1.7	23 21 29		(24) 4.2	33 30 25	(29) 4.0	11 16 16	(14) 2.9
+	5000	158 P 177 P 167 P	(167) 9.5	13 P 18 P 10 P	(14) 4.0	23 P 21 P 21 P		(22) 1.2	31 P 24 P 33 P	(29) 4.7	12 P 13 P 15 P	(13) 1.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1062 962 874	(966) 94.1	163 172 186	(174) 11.6	377 330 318		(342) 31.2	212 172 175	(186) 22.3	343 401 385	(376) 30.0

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

#        Standard deviation Table4              Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 20 April 2010					To: 23 April 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	168 156 159	(161) 6.2#	26 24 23	(24) 1.5	23 21 18		(21) 2.5	26 29 30	(28) 2.1	9 13 8	(10) 2.6
-	50	154 136 152	(147) 9.9	21 24 29	(25) 4.0	21 24 14		(20) 5.1	25 29 31	(28) 3.1	9 10 11	(10) 1.0
-	150	151 142 144	(146) 4.7	21 26 29	(25) 4.0	25 20 15		(20) 5.0	20 27 26	(24) 3.8	11 11 7	(10) 2.3
-	500	151 158 141	(150) 8.5	26 23 25	(25) 1.5	19 16 14		(16) 2.5	24 26 25	(25) 1.0	9 11 10	(10) 1.0
-	1500	155 156 151	(154) 2.6	23 27 22	(24) 2.6	20 18 21		(20) 1.5	26 20 26	(24) 3.5	12 9 10	(10) 1.5
-	5000	156 P 152 P 139 P	(149) 8.9	27 P 25 P 26 P	(26) 1.0	20 P 21 P 22 P		(21) 1.0	18 P 22 P 22 P	(21) 2.3	10 P 8 P 8 P	(9) 1.2
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		407 420 368	(398) 27.1	236 244 172	(217) 39.5	363 387 423		(391) 30.2	663 510 291	(488) 187.0	2374 2028 2084	(2162) 185.7

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

#        Standard deviation Table5              Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 20 April 2010					To: 23 April 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	173 159 157	(163) 8.7#	16 20 16	(17) 2.3	32 33 22		(29) 6.1	31 34 30	(32) 2.1	12 16 14	(14) 2.0
+	50	154 178 143	(158) 17.9	16 12 14	(14) 2.0	31 20 18		(23) 7.0	31 27 31	(30) 2.3	8 12 7	(9) 2.6
+	150	144 164 141	(150) 12.5	14 18 13	(15) 2.6	20 23 19		(21) 2.1	28 34 29	(30) 3.2	15 10 13	(13) 2.5
+	500	162 158 163	(161) 2.6	16 11 13	(13) 2.5	30 33 26		(30) 3.5	25 31 29	(28) 3.1	12 14 8	(11) 3.1
+	1500	194 147 184	(175) 24.8	13 13 14	(13) 0.6	27 23 26		(25) 2.1	31 26 33	(30) 3.6	15 14 13	(14) 1.0
+	5000	168 P 174 P 135 P	(159) 21.0	12 P 17 P 16 P	(15) 2.6	29 P 26 P 23 P		(26) 3.0	32 P 34 P 29 P	(32) 2.5	14 P 13 P 15 P	(14) 1.0
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1398 1669 1671	(1579) 157.0	329 311 354	(331) 21.6	187 219 199		(202) 16.2	235 202 268	(235) 33.0	173 243 291	(236) 59.3

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

#        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.