Synthetic wollastonite

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:

06 January 2021 - 01 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Synthetic wollastonite

EC Number:

952-026-5

Molecular formula:

CaSiO3

IUPAC Name:

Synthetic wollastonite

Test material form:

solid: bulk

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Imerys
- Lot/batch number of test material: AGRG-00620
- Purity, including information on contaminants, isomers, etc.: 100 %
- Molecular weight: 116.16 g/mol
- Expiry date: 27 November 2030

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 insoluble in sterile distilled water. The test item formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987 and Trinova Biochem GmbH on 27 June 2017
- Suitability of cells:
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: Frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-; Base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally, due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000).

For cell lines:
- Methods for maintenance in cell culture: All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.On a regular basis (approximately monthly), batches of culture from master stocks are prepared and coded, these are then routinely tested for appropriate characteristics, viability and mutation frequency to ensure acceptability criteria is met.

MEDIA USED
- Type and composition of media: Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 2928960 expiry date 02/2025) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Species / strain 2

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987 and Trinova Biochem GmbH on 27 June 2017
- Suitability of cells: Genotype: trp-; uvrA-:
Type of mutation: Base-pair substitution
In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

For cell lines:
- Methods for maintenance in cell culture: All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.On a regular basis (approximately monthly), batches of culture from master stocks are prepared and coded, these are then routinely tested for appropriate characteristics, viability and mutation frequency to ensure acceptability criteria is met.

MEDIA USED
- Type and composition of media: Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 2928960 expiry date 02/2025) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Phenobarbitone / β-Naphthoflavone induced Microsomal fractions (Sprague-Dawley)

- source of S9: Moltox; Lot No.’s 4272 (Experiment 1), 4217 (Experiment 2) and 4370 (Experiment 2 repeat) with the protein level adjusted to 20 mg/mL.

- method of preparation of S9 mix: The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

S9 fraction 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

- quality controls of S9: A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine (for S. typhimurium strains) or tryptophan (for E.coli strain) supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate
Experiment 2: 15, 50, 150, 500, 1500 and 5000 ug/plate
Selected based on the lack of cytotoxicity noted in Experiment 1.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- 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, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected.

- Justification for percentage of solvent in the final culture medium: The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in high purity DMSO by mixing on a vortex mixer and sonication for 20 minutes at 40 °C. No correction for purity was required. All test item preparation and dosing was performed under yellow safety lighting.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

Presence of S9-mix.

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

In the absence of S9 mix.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate

METHOD OF TREATMENT/ EXPOSURE:
Experiment 1
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel Bonner agar plate.
With Metabolic Activation
The procedure was the same as described above except that untreated controls were not performed and, following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

TREATMENT AND HARVEST SCHEDULE:
All of the plates were incubated at 37 ± 3 Deg C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity).

METHOD OF TREATMENT/ EXPOSURE:
Experiment 2
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent or 0.1 mL of appropriate positive control were incubated at 37 ± 3 Deg C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates.
With Metabolic Activation
The procedure was the same as described above except that untreated controls were not performed and, following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 Deg C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media.

TREATMENT AND HARVEST SCHEDULE:
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
All of the plates were incubated at 37 ± 3 Deg C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity). Manual counts were performed at 5000 ug/plate (where applicable) because of test item precipitation. A further manual count was performed for TA1537 at 5000 µg/plate as different sized colonies prevented an accurate automated count.

Rationale for test conditions:

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), Green and Muriel (1976), and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the solvent and untreated controls. Typical published ranges are presented as follows:
Strain TA1535 7 7 to 40
Strain TA100 60 to 200
Strain TA1537 2 to 30
Strain TA98 8 to 60
Strain WP2uvrA 10 to 60
These values were confirmed against current in-house historical control profiles to further validate acceptability. Although the number of spontaneous revertants can be expected to fall within the ranges, they may occasionally fall outside these.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation (S9-mix).
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

Statistical significance was not included as part of the result evaluation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A test item precipitate (white and granular in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix (Experiment 2). The precipitate did not prevent the scoring of revertant colonies.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: negative

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Results for the untreated controls (spontaneous mutation rates) and viability were considered to be acceptable. These data are for concurrent untreated control plates dosed in the absence of S9 performed on the same day as the Mutation Test.
The number of revertant counts for the solvent (dimethyl sulphoxide) control plates were 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 and without S9 mix. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

For all test methods and criteria for data analysis and interpretation:
Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all checks were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.
Ames test:
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and solvent controls, both with and without S9-mix, are presented in Table 1 and Table 2 for Experiment 1 and Table 3 and Table 4 for Experiment 2 (see "Any other information on results incl. tables" below for details).

Any other information on results incl. tables

Experiment 1 (plate incorporation) – Table 1 and Table 2

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix.

No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix. 

Table 1 Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From:26 January 2021 29 January 2021†					To: 29 January 2021 01 February 2021†
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA†			TA98		TA1537
	Solvent Control DMSO	125 139 128	(131) 7.4#	7 9 16	(11) 4.7	16 20 24		(20) 4.0	20 11 22	(18) 5.9	10 9 11	(10) 1.0
	1.5 µg	110 131 153	(131) 21.5	10 16 10	(12) 3.5	18 26 17		(20) 4.9	21 21 14	(19) 4.0	14 11 15	(13) 2.1
	5 µg	119 118 133	(123) 8.4	9 16 15	(13) 3.8	21 12 22		(18) 5.5	19 23 12	(18) 5.6	14 17 17	(16) 1.7
	15 µg	114 114 144	(124) 17.3	12 11 11	(11) 0.6	15 18 16		(16) 1.5	13 17 18	(16) 2.6	8 11 11	(10) 1.7
	50 µg	113 131 123	(122) 9.0	11 11 13	(12) 1.2	26 21 17		(21) 4.5	15 23 16	(18) 4.4	13 13 10	(12) 1.7
	150 µg	135 126 121	(127) 7.1	16 11 12	(13) 2.6	14 14 27		(18) 7.5	13 17 13	(14) 2.3	7 17 11	(12) 5.0
	500 µg	120 131 131	(127) 6.4	9 17 7	(11) 5.3	23 13 19		(18) 5.0	19 17 16	(17) 1.5	15 7 12	(11) 4.0
	1500 µg	112 114 114	(113) 1.2	2 17 12	(10) 7.6	13 19 20		(17) 3.8	18 27 10	(18) 8.5	18 9 9	(12) 5.2
	5000 µg	120 138 129	(129) 9.0	14 13 14	(14) 0.6	21 22 21		(21) 0.6	19 19 15	(18) 2.3	15 12 10	(12) 2.5
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	535 493 544	(524) 27.2	977 903 1324	(1068) 224.8	740 824 741		(768) 48.2	118 126 129	(124) 5.7	161 163 304	(209) 82.0

 

†                 Experimental procedure repeated at a later date due to lack of colony growth

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

4NQO       4-Nitroquinoline-1-oxide

9AA          9-Aminoacridine

#          Standard deviation

Table 2     Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From:26 January 2021 29 January 2021†					To: 29 January 2021 01 February 2021†
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA†			TA98		TA1537
	Solvent Control DMSO	114 137 116	(122) 12.7#	8 13 12	(11) 2.6	25 21 22		(23) 2.1	28 25 20	(24) 4.0	12 10 10	(11) 1.2
	1.5 µg	129 114 185	(143) 37.4	13 14 10	(12) 2.1	18 22 21		(20) 2.1	29 14 22	(22) 7.5	12 10 19	(14) 4.7
	5 µg	109 128 106	(114) 11.9	10 12 5	(9) 3.6	24 16 25		(22) 4.9	28 18 27	(24) 5.5	23 18 6	(16) 8.7
	15 µg	128 116 131	(125) 7.9	12 10 8	(10) 2.0	24 23 33		(27) 5.5	22 27 24	(24) 2.5	12 6 15	(11) 4.6
	50 µg	106 121 137	(121) 15.5	10 6 7	(8) 2.1	27 22 13		(21) 7.1	22 23 33	(26) 6.1	17 8 12	(12) 4.5
	150 µg	133 111 137	(127) 14.0	6 8 12	(9) 3.1	17 21 18		(19) 2.1	25 14 23	(21) 5.9	11 18 15	(15) 3.5
	500 µg	126 148 160	(145) 17.2	15 5 10	(10) 5.0	19 20 14		(18) 3.2	24 32 24	(27) 4.6	10 7 10	(9) 1.7
	1500 µg	120 139 139	(133) 11.0	13 14 14	(14) 0.6	27 19 22		(23) 4.0	33 28 22	(28) 5.5	14 9 14	(12) 2.9
	5000 µg	121 143 128	(131) 11.2	11 7 14	(11) 3.5	23 21 22		(22) 1.0	25 33 19	(26) 7.0	9 13 9	(10) 2.3
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	2774 2647 2692	(2704) 64.4	380 390 355	(375) 18.0	197 186 235		(206) 25.7	163 149 167	(160) 9.5	324 324 332	(327) 4.6

 

†             Experimental procedure repeated at a later date due to lack of colony growth

BP          Benzo(a)pyrene

2AA       2-Aminoanthracene

#          Standard deviation

 

Experiment 2 (pre-incubation) – Table 3 and Table 4

A visible reduction in the growth of the bacterial background lawn was noted at 5000 µg/plate for all of the tester strains dosed in the absence of S9-mix after employing the pre-incubation modification.  However, there was no visible reduction in the growth of the bacterial background lawn at any dose level, in the presence of S9-mix.

A test item precipitate (white and granular in appearance) was noted at and above 500 mg/plate in both the presence and absence of S9-mix in Experiment 2 after performing the pre-incubation method.  The precipitate did not prevent the scoring of revertant colonies.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

Table 3             Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From:19 February 2021 25 February 2021†					To: 22 February 2021 28 February 2021†
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535 †		WP2uvrA			TA98 †		TA1537
	Solvent Control DMSO	160 152 155	(156) 4.0#	21 17 11	(16) 5.0	15 15 25		(18) 5.8	17 19 15	(17) 2.0	16 11 11	(13) 2.9
	15 µg	164 158 176	(166) 9.2	16 15 10	(14) 3.2	25 19 20		(21) 3.2	20 19 18	(19) 1.0	19 12 12	(14) 4.0
	50 µg	157 173 175	(168) 9.9	13 16 14	(14) 1.5	19 22 14		(18) 4.0	10 28 19	(19) 9.0	13 13 19	(15) 3.5
	150 µg	176 168 164	(169) 6.1	12 14 15	(14) 1.5	18 29 15		(21) 7.4	16 13 17	(15) 2.1	14 14 20	(16) 3.5
	500 µg	177 P 170 P 169 P	(172) 4.4	21 P 17 P 18 P	(19) 2.1	22 P 25 P 25 P		(24) 1.7	18 P 17 P 15 P	(17) 1.5	11 P 9 P 11 P	(10) 1.2
	1500 µg	146 P 180 P 152 P	(159) 18.1	18 P 19 P 14 P	(17) 2.6	29 P 16 P 19 P		(21) 6.8	19 P 20 P 20 P	(20) 0.6	14 P 12 P 12 P	(13) 1.2
	5000 µg	158 PS 176 PS 174 PS	(169) 9.9	12 PS 16 PS 18 PS	(15) 3.1	22 PS 34 PS 30 PS		(29) 6.1	15 PS 11 PS 17 PS	(14) 3.1	14 P S 21 P S 14 P S	(16) 4.0
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	1053 1028 1071	(1051) 21.6	1158 847 1196	(1067) 191.5	898 884 821		(868) 41.0	249 254 285	(263) 19.5	158 407 420	(328) 147.7

 

†                 Experimental procedure repeated at a later date due to contamination

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

4NQO       4-Nitroquinoline-1-oxide

9AA          9-Aminoacridine

P                Test item precipitate

S                Sparse bacterial background lawn

#                        Standard deviation

 

Table 4             Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From:19 February 2021 25 February 2021†					To: 22 February 2021 28 February 2021†
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535 †		WP2uvrA			TA98 †		TA1537
	Solvent Control DMSO	163 170 184	(172) 10.7#	19 15 14	(16) 2.6	37 38 29		(35) 4.9	25 19 25	(23) 3.5	15 12 24	(17) 6.2
	15 µg	147 168 153	(156) 10.8	21 7 14	(14) 7.0	46 39 31		(39) 7.5	24 20 17	(20) 3.5	18 10 9	(12) 4.9
	50 µg	160 163 157	(160) 3.0	16 13 14	(14) 1.5	36 29 24		(30) 6.0	17 23 22	(21) 3.2	12 10 14	(12) 2.0
	150 µg	141 152 151	(148) 6.1	14 14 14	(14) 0.0	27 29 17		(24) 6.4	23 19 24	(22) 2.6	15 20 12	(16) 4.0
	500 µg	132 P 142 P 159 P	(144) 13.7	10 P 10 P 14 P	(11) 2.3	22 P 37 P 28 P		(29) 7.5	20 P 23 P 24 P	(22) 2.1	12 P 17 P 16 P	(15) 2.6
	1500 µg	141 P 154 P 134 P	(143) 10.1	10 P 11 P 15 P	(12) 2.6	33 P 33 P 32 P		(33) 0.6	16 P 28 P 19 P	(21) 6.2	12 P 23 P 10 P	(15) 7.0
	5000 µg	162 P 143 P 132 P	(146) 15.2	11 P 13 P 12 P	(12) 1.0	23 P 20 P 36 P		(26) 8.5	22 P 16 P 15 P	(18) 3.8	10 P 20 P 21 P	(17) 6.1
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	2691 2540 2739	(2657) 103.8	298 245 264	(269) 26.9	136 157 154		(149) 11.4	166 172 140	(159) 17.0	430 438 426	(431) 6.1

 

†             Experimental procedure repeated at a later date due to contamination

BP          Benzo(a)pyrene

2AA       2-Aminoanthracene

P             Test item precipitate

#                        Standard deviation

 

 

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Synthetic Wollastonite did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test Synthetic Wollastonite was considered to be non-mutagenic.