Synthetic wollastonite

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames study carried out on Synthetic wollastonite gave a negative result for mutagenicity with no indication of cytotoxicity. 

An Ames study carried out on the analogue substance Kieselguhr soda ash flux also gave a negative result for mutagenicity with no indication of cytotoxicity.

A Chromosome aberration study carried out on the read-across test material Kieselguhr soda ash flux calcined was negative for cytogenicity.

A gene mutation study carried out on the read-across test material Kieselguhr soda ash fluc calcined was found to be negative for mutagenicity.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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.

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 / 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.

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.

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.

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

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

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

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

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

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

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

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

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

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).

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

 

 

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 22 February 2010 and 30 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal calf serum, at approximately 37ºC with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at approximately 90 µg/ml final concentration

Metabolic activation:

with and without

Metabolic activation system:

Induced rat liver homogenate metabolising system (S9)

Test concentrations with justification for top dose:

Preliminary expertiment: 2.34, 4.69, 9.38, 18.75, 37.5, 75, 150, 300 and 600 µg/mL.

Main experiment: 1.25 , 2.5, 5, 10, 20 and 40 µg/mL:

Negative solvent / vehicle controls:

yes

Remarks:

Minimal essential medium

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: In the absence of S9, used at 0.4 and 0.2 µg/mL for the 4(20)-hour and 24-hour exposure groups respectively

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulphoxide

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: In the presence of S9, used at 5 µg/mL

Details on test system and experimental conditions:

DURATION:
Preliminary toxicity test:
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation

Main experiment:
4-hour exposure to the test material without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.
4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.
24-hour continuous exposure to the test material without S9-mix prior to cell harvest.

STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.


NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted

DETERMINATION OF CYTOTOXICITY
- Method: The number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 2.34 to 600 µg/mL. The maximum dose was based on the maximum recommended 10 mM concentration. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 37.5 µg/mL in the 4(20)-hour exposure groups and at and above 9.38 µg/mL in the 24-hour continuous exposure group. Precipitate was also carried through onto the slides of the 24-hour exposure group at 300 and 600 µg/mL. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 600 µg/mL in all three exposure groups. The mitotic index data are presented in Table 1. The test material induced no evidence of toxicity in any of the exposure groups. There was a slight reduction in mitotic index observed in the 4(20)-hour exposure group in the presence of S9 but this was considered to be as a result of a particularly high vehicle control value.
The selection of the maximum dose level for the exposure groups of the main experiment was based on the lowest precipitating dose level observed in the 4(20)-hour exposure groups of the preliminary toxicity test and was 40 µg/mL for all three exposure groups.


CHROMOSOME ABERRATION TEST:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 40 µg/mL in all three exposure groups. Precipitate was not seen in the blood cultures of this experiment but this was considered to be due to the masking effects of the blood and the observations taken in the media only tubes of the Preliminary Toxicity Test are considered to be representative for the study.

The mitotic index data are given in Table 2 and Table 3. They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the exposure groups of the main experiment.

The maximum dose level selected for metaphase analysis was 40 µg/mL.

The chromosome aberration data are given in Table 4, Table 5 and Table 6. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 7. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

There was no evidence of a response in the presence of metabolic activation in this study or in the MLA test performed on the test material (Harlan Project No.3018/0010). This was taken as scientific justification to confirm that the repeat of the 4(20)-hour exposure group with metabolic action was not required

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Mitotic index - Preliminary toxicity test

4-HOUR TREATMENT, 20-HOUR RECOVERY -S9
4-HOUR TREATMENT, 20-HOUR RECOVERY +S9
24-HOUR TREATMENT -S9
CONCENTRATION (µg/ml)	4(20)h WITHOUT S9		4(20)h WITH S9		24h WITHOUT S9
	MITOTIC INDEX	% OF CONTROL	MITOTIC INDEX	% OF CONTROL	MITOTIC INDEX	% OF CONTROL
0	7.55	100	9.40	100	9.05	100
2.34	-	-	-	-	-	-
4.69	-	-	-	-	7.55	83
9.38	-	-	-	-	7.75 P	86
18.75	8.25	109	5.90	63	8.75 P	97
37.5	7.65 P	101	7.20 P	77	6.45 P	71
75	9.50 P	126	8.30 P	88	- P	-
150	8.05 P	107	7.55 P	80	- P	-
300	- P	-	- P	-	- P	-
600	8.75 P	116	7.15 P	76	7.90 P	87

 - = Not assessed for mitotic index     

P = Precipitate

Table 2: Mitotic index - 4(20) - hours

DOSE LEVEL µg/ml	4 HOURS TREATMENT WITHOUT S9				4 HOURS TREATMENT WITH S9
	A	B	MEAN	% OF CONTROL	A	B	MEAN	% OF CONTROL
0	5.85	5.10	5.48	100	4.50	4.95	4.73	100
1.25	-	-	-	-	-	-	-	-
2.5	-	-	-	-	-	-	-	-
5	-	-	-	-	-	-	-	-
10	5.55	6.80	6.18	113	6.05	5.75	5.90	125
20	5.40	6.20	5.80	106	4.90	6.45	5.68	120
40	6.10	6.55	6.33	116	5.40	5.15	5.28	112
MMC 0.4	3.40	2.70	3.05	56	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	1.65	1.55	1.60	34

MMC  = Mitomycin C

CP  = Cyclophosphamide

NA  = Not applicable

-    = Not assessed for mitotic index Table 3: Mitotic index - 24 hours

DOSE LEVEL µg/ml	24 HOURS TREATMENT WITHOUT S9
	A	B	MEAN	% OF CONTROL
0	6.70	6.40	6.55	100
1.25	-	-	-	-
2.5	-	-	-	-
5	-	-	-	-
10	6.25	5.65	5.95	91
20	4.80	6.10	5.45	83
40	6.95	6.45	6.70	102
MMC 0.2	1.80	1.85	1.83	28

MMC  = Mitomycin C               

- = Not assessed for mitotic index

Table 4: Results of chromosome aberration test - 4(20) hours without metabolic activation (S9)

Treatment group	Replicate	Mitotic index (%)	Number of cells scored	Number of aberrations						Total number of aberrations		Frequency of aberrant cells (%)
				Gaps	Chromatid		Chromosome		Others	( + gaps)	( - gaps)	( + gaps)	( - gaps)
					Breaks	Exchanges	Breaks	Exchanges	X
Vehicle control (MEM)	A	5.85	100	0	0	0	0	0	0	0	0	0	0
	B	5.10	100	2	1	0	6	0	0	9	7	4	2
	Total		200	2	1	0	6	0	0	9	7	4	2
		(100)										(2.0)	(1.0)
10 µg/mL	A		100	0	0	1	0	0	0	1	1	1	1
	B		100	1	0	0	0	0	0	1	0	1	0
	Total		200	1	0	1	0	0	0	2	1	2	1
		(113)										(1.0)	(0.5)
20 µg/mL	A		100	0	0	0	1	0	0	1	1	1	1
	B		100	0	3	1	0	0	0	4	4	4	4
	Total		200	0	3	1	1	0	0	5	5	5	5
		(106)										(2.5)	(2.5)
40 µg/mL	A	6.10	100	0	0	2	0	0	0	2	2	1	1
	B	6.55	100	0	0	0	0	0	0	0	0	0	0
	Total		200	0	0	2	0	0	0	2	2	1	1
		(116)										(0.5)	(0.5)
Positive control MMC 0.4µg/mL	A	3.40	50^a	8	21	18	2	0	1	50	42	27	25
	B	2.70	50^a	16	15	20	5	0	0	56	40	27	33
	Total		100	24	36	38	7	0	1	106	82	54	48***
		(56)										(54.0)	(48.0)

MMC= Mitomycin C

a     = Slide evaluation terminated at 50 cells because approximately 30% to 50% cells with aberrations had been observed

***   = P < 0.001

MEM= Minimal Essential Medium

Conclusions:

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 22 February 2010 and 30 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

The use of data derived for Soda-ash flux calcined kieselghur are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal calf serum, at approximately 37ºC with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at approximately 90 µg/ml final concentration

Metabolic activation:

with and without

Metabolic activation system:

Induced rat liver homogenate metabolising system (S9)

Test concentrations with justification for top dose:

Preliminary expertiment: 2.34, 4.69, 9.38, 18.75, 37.5, 75, 150, 300 and 600 µg/mL.

Main experiment: 1.25 , 2.5, 5, 10, 20 and 40 µg/mL:

Negative solvent / vehicle controls:

yes

Remarks:

Minimal essential medium

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: In the absence of S9, used at 0.4 and 0.2 µg/mL for the 4(20)-hour and 24-hour exposure groups respectively

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulphoxide

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: In the presence of S9, used at 5 µg/mL

Details on test system and experimental conditions:

DURATION:
Preliminary toxicity test:
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation

Main experiment:
4-hour exposure to the test material without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.
4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.
24-hour continuous exposure to the test material without S9-mix prior to cell harvest.

STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.


NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted

DETERMINATION OF CYTOTOXICITY
- Method: The number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 2.34 to 600 µg/mL. The maximum dose was based on the maximum recommended 10 mM concentration. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 37.5 µg/mL in the 4(20)-hour exposure groups and at and above 9.38 µg/mL in the 24-hour continuous exposure group. Precipitate was also carried through onto the slides of the 24-hour exposure group at 300 and 600 µg/mL. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 600 µg/mL in all three exposure groups. The mitotic index data are presented in Table 1. The test material induced no evidence of toxicity in any of the exposure groups. There was a slight reduction in mitotic index observed in the 4(20)-hour exposure group in the presence of S9 but this was considered to be as a result of a particularly high vehicle control value.
The selection of the maximum dose level for the exposure groups of the main experiment was based on the lowest precipitating dose level observed in the 4(20)-hour exposure groups of the preliminary toxicity test and was 40 µg/mL for all three exposure groups.


CHROMOSOME ABERRATION TEST:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 40 µg/mL in all three exposure groups. Precipitate was not seen in the blood cultures of this experiment but this was considered to be due to the masking effects of the blood and the observations taken in the media only tubes of the Preliminary Toxicity Test are considered to be representative for the study.

The mitotic index data are given in Table 2 and Table 3. They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the exposure groups of the main experiment.

The maximum dose level selected for metaphase analysis was 40 µg/mL.

The chromosome aberration data are given in Table 4, Table 5 and Table 6. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 7. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

There was no evidence of a response in the presence of metabolic activation in this study or in the MLA test performed on the test material (Harlan Project No.3018/0010). This was taken as scientific justification to confirm that the repeat of the 4(20)-hour exposure group with metabolic action was not required

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Mitotic index - Preliminary toxicity test

4-HOUR TREATMENT, 20-HOUR RECOVERY -S9
4-HOUR TREATMENT, 20-HOUR RECOVERY +S9
24-HOUR TREATMENT -S9
CONCENTRATION (µg/ml)	4(20)h WITHOUT S9		4(20)h WITH S9		24h WITHOUT S9
	MITOTIC INDEX	% OF CONTROL	MITOTIC INDEX	% OF CONTROL	MITOTIC INDEX	% OF CONTROL
0	7.55	100	9.40	100	9.05	100
2.34	-	-	-	-	-	-
4.69	-	-	-	-	7.55	83
9.38	-	-	-	-	7.75 P	86
18.75	8.25	109	5.90	63	8.75 P	97
37.5	7.65 P	101	7.20 P	77	6.45 P	71
75	9.50 P	126	8.30 P	88	- P	-
150	8.05 P	107	7.55 P	80	- P	-
300	- P	-	- P	-	- P	-
600	8.75 P	116	7.15 P	76	7.90 P	87

 - = Not assessed for mitotic index     

P = Precipitate

Table 2: Mitotic index - 4(20) - hours

DOSE LEVEL µg/ml	4 HOURS TREATMENT WITHOUT S9				4 HOURS TREATMENT WITH S9
	A	B	MEAN	% OF CONTROL	A	B	MEAN	% OF CONTROL
0	5.85	5.10	5.48	100	4.50	4.95	4.73	100
1.25	-	-	-	-	-	-	-	-
2.5	-	-	-	-	-	-	-	-
5	-	-	-	-	-	-	-	-
10	5.55	6.80	6.18	113	6.05	5.75	5.90	125
20	5.40	6.20	5.80	106	4.90	6.45	5.68	120
40	6.10	6.55	6.33	116	5.40	5.15	5.28	112
MMC 0.4	3.40	2.70	3.05	56	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	1.65	1.55	1.60	34

MMC  = Mitomycin C

CP  = Cyclophosphamide

NA  = Not applicable

-    = Not assessed for mitotic index Table 3: Mitotic index - 24 hours

DOSE LEVEL µg/ml	24 HOURS TREATMENT WITHOUT S9
	A	B	MEAN	% OF CONTROL
0	6.70	6.40	6.55	100
1.25	-	-	-	-
2.5	-	-	-	-
5	-	-	-	-
10	6.25	5.65	5.95	91
20	4.80	6.10	5.45	83
40	6.95	6.45	6.70	102
MMC 0.2	1.80	1.85	1.83	28

MMC  = Mitomycin C               

- = Not assessed for mitotic index

Table 4: Results of chromosome aberration test - 4(20) hours without metabolic activation (S9)

Treatment group	Replicate	Mitotic index (%)	Number of cells scored	Number of aberrations						Total number of aberrations		Frequency of aberrant cells (%)
				Gaps	Chromatid		Chromosome		Others	( + gaps)	( - gaps)	( + gaps)	( - gaps)
					Breaks	Exchanges	Breaks	Exchanges	X
Vehicle control (MEM)	A	5.85	100	0	0	0	0	0	0	0	0	0	0
	B	5.10	100	2	1	0	6	0	0	9	7	4	2
	Total		200	2	1	0	6	0	0	9	7	4	2
		(100)										(2.0)	(1.0)
10 µg/mL	A		100	0	0	1	0	0	0	1	1	1	1
	B		100	1	0	0	0	0	0	1	0	1	0
	Total		200	1	0	1	0	0	0	2	1	2	1
		(113)										(1.0)	(0.5)
20 µg/mL	A		100	0	0	0	1	0	0	1	1	1	1
	B		100	0	3	1	0	0	0	4	4	4	4
	Total		200	0	3	1	1	0	0	5	5	5	5
		(106)										(2.5)	(2.5)
40 µg/mL	A	6.10	100	0	0	2	0	0	0	2	2	1	1
	B	6.55	100	0	0	0	0	0	0	0	0	0	0
	Total		200	0	0	2	0	0	0	2	2	1	1
		(116)										(0.5)	(0.5)
Positive control MMC 0.4µg/mL	A	3.40	50^a	8	21	18	2	0	1	50	42	27	25
	B	2.70	50^a	16	15	20	5	0	0	56	40	27	33
	Total		100	24	36	38	7	0	1	106	82	54	48***
		(56)										(54.0)	(48.0)

MMC= Mitomycin C

a     = Slide evaluation terminated at 50 cells because approximately 30% to 50% cells with aberrations had been observed

***   = P < 0.001

MEM= Minimal Essential Medium

Conclusions:

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The study was performed between 25 February 2010 and 23 March 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

The use of data derived for Soda-ash flux calcined kieselghur are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Prior to freezing stocks of the cells, they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours.

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal enzyme fraction

Test concentrations with justification for top dose:

Preliminary toxicity test:0, 2.34, 4.69, 9.38, 18.75, 37.5, 75, 150, 300 and 600 µg/L

Main test: 0, 2.5, 5, 10, 20, 30, and 40μg/mL for all three of the exposure groups

Vehicle / solvent:

- Vehicle used: R0 medium

Negative solvent / vehicle controls:

yes

Remarks:

R0 medium

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: At 400 µg/mL and 150 µg/mL for the 4 hour and 24-hour exposures respectively in the absence of metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

R0 medium

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: At 2 µg/mL in the presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: in Experiment 1, cells were treated with the test material for 4 hours both in the presence and absence of metabolic activation. In Experiment 2, cells were treated with the test material for 4 hours in the presence of metabolic activation and 24 hours in the absence of metabolic activation.
- Expression time : 2 days
- Selection time : up to 48 hours

SELECTION AGENT :5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: performed in duplicate (denoted as A and B)

NUMBER OF CELLS EVALUATED: 10^4 cells/mL (2000 cells/well) plated in selective medium for evaluation of mutant frequency and 10 cells/mL (2 cells/well) to assess viability (%V) plated in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: MTT (2.5 mg/mL in PBS) was added to each well of the mutation plates to aid in counting viable cells.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10-6 mutant frequency per survivor are not normally acceptable and will be repeated


Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

During the course of the study dose selection for the mutagenicity experiments will be made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Both %RSG and RTG values are used either individually or combined to designate the level of toxicity achieved by the test material for any individual dose level. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.

Statistics:

The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Preliminary toxicity test

Dose (µg/ml)	%(-S9) 4-Hour Exposure	%(+S9) 4-Hour Exposure	%(-S9) 24-Hour Exposure
0	100	100	100
2.34	109	97	84
4.69	101	96	85
9.38	107	95	92
18.75	95	101	74
37.5	90	94	76
75	107	85	91
150	91	95	84
300	102	86	89
600	111	97	104

Table 2: Results from main experiment

Treatment (µg/mL)	4 hours - S9			Treatment (µg/mL)	4 hours + S9
	% RSG	RTG	MF§		% RSG	RTG	MF§
0	100	1.00	88.96	0	100	1.00	60.42
2.5	113	1.15	80.91	2.5	109	1.00	81.64
5	108	1.11	88.25	5	114	1.14	69.10
10	110	1.01	92.35	10	125	1.15	63.40
20	96	1.05	91.19	20	115	1.06	74.48
30	117	1.13	125.24	30	128	1.06	77.99
40	103	1.02	80.50	40	121	0.92	122.02*
Linear trend                                      NS				Linear trend                                      *
				CP
400	76	0.53	1060.81	2	68	0.33	1112.22

Treatment (µg/mL)	24 hours S9
	% RSG	RTG	MF§
0	100	1.00	73.91
2.5	102	1.22	67.81
5	89	1.21	69.85
10	105	1.27	84.26
20	97	1.20	69.77
30	102	1.15	61.92
40	103	1.30	53.05
Linear trend                                      NS
150	57	0.38	741.41

Key to tables:

% RSG = Relative suspension growth

RTG = Relative Total Growth

MF § = 5 -TFT resistant mutants/10⁶ viable cells 2 days after treatment

* = p<0.05

NS = Not significant

Conclusions:

Negative in the presence and absence of metabolic activation

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 25 February 2010 and 23 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Prior to freezing stocks of the cells, they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours.

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal enzyme fraction

Test concentrations with justification for top dose:

Preliminary toxicity test:0, 2.34, 4.69, 9.38, 18.75, 37.5, 75, 150, 300 and 600 µg/L

Main test: 0, 2.5, 5, 10, 20, 30, and 40μg/mL for all three of the exposure groups

Vehicle / solvent:

- Vehicle used: R0 medium

Negative solvent / vehicle controls:

yes

Remarks:

R0 medium

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: At 400 µg/mL and 150 µg/mL for the 4 hour and 24-hour exposures respectively in the absence of metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

R0 medium

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: At 2 µg/mL in the presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: in Experiment 1, cells were treated with the test material for 4 hours both in the presence and absence of metabolic activation. In Experiment 2, cells were treated with the test material for 4 hours in the presence of metabolic activation and 24 hours in the absence of metabolic activation.
- Expression time : 2 days
- Selection time : up to 48 hours

SELECTION AGENT :5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: performed in duplicate (denoted as A and B)

NUMBER OF CELLS EVALUATED: 10^4 cells/mL (2000 cells/well) plated in selective medium for evaluation of mutant frequency and 10 cells/mL (2 cells/well) to assess viability (%V) plated in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: MTT (2.5 mg/mL in PBS) was added to each well of the mutation plates to aid in counting viable cells.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10-6 mutant frequency per survivor are not normally acceptable and will be repeated


Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

During the course of the study dose selection for the mutagenicity experiments will be made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Both %RSG and RTG values are used either individually or combined to designate the level of toxicity achieved by the test material for any individual dose level. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.

Statistics:

The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Preliminary toxicity test

Dose (µg/ml)	%(-S9) 4-Hour Exposure	%(+S9) 4-Hour Exposure	%(-S9) 24-Hour Exposure
0	100	100	100
2.34	109	97	84
4.69	101	96	85
9.38	107	95	92
18.75	95	101	74
37.5	90	94	76
75	107	85	91
150	91	95	84
300	102	86	89
600	111	97	104

Table 2: Results from main experiment

Treatment (µg/mL)	4 hours - S9			Treatment (µg/mL)	4 hours + S9
	% RSG	RTG	MF§		% RSG	RTG	MF§
0	100	1.00	88.96	0	100	1.00	60.42
2.5	113	1.15	80.91	2.5	109	1.00	81.64
5	108	1.11	88.25	5	114	1.14	69.10
10	110	1.01	92.35	10	125	1.15	63.40
20	96	1.05	91.19	20	115	1.06	74.48
30	117	1.13	125.24	30	128	1.06	77.99
40	103	1.02	80.50	40	121	0.92	122.02*
Linear trend                                      NS				Linear trend                                      *
				CP
400	76	0.53	1060.81	2	68	0.33	1112.22

Treatment (µg/mL)	24 hours S9
	% RSG	RTG	MF§
0	100	1.00	73.91
2.5	102	1.22	67.81
5	89	1.21	69.85
10	105	1.27	84.26
20	97	1.20	69.77
30	102	1.15	61.92
40	103	1.30	53.05
Linear trend                                      NS
150	57	0.38	741.41

Key to tables:

% RSG = Relative suspension growth

RTG = Relative Total Growth

MF § = 5 -TFT resistant mutants/10⁶ viable cells 2 days after treatment

* = p<0.05

NS = Not significant

Conclusions:

Negative in the presence and absence of metabolic activation

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Study was performed between 25 February 2010 and 23 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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.

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

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:

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:

80 μg/plate for TA1537 - without S9-mix

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

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:

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.

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 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'.

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

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Information from three in vivo studies carried out on the read across test material calcium oxosilanediolate (silicic acid, calcium salt) is available. 

A Chromosome aberration study carried out in the rat concluded that calcium oxosilanediolate was negative for cytogenicity.

A Dominant Lethal Assay carried out in the rat concluded that calcium oxosilanediolate was considered to be non-mutagenic.

A gene mutation study carried our in the mouse concluded that calcium oxosilanediolate was considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline available

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Principles of method if other than guideline:

Analysis of metaphase-chromosomes from isolated bone-marrow cells after colcemid-induced arrest of cell division in the metaphase

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: 10 - 12 weeks

Route of administration:

oral: gavage

Vehicle:

- vehicle: 0.85 % saline
- Volume of the vehicle: no data
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data


MAXIMUM DOSE VOLUME APPLIED: no data

Details on exposure:

3 time points: sampling at 6, 24, and 48 h in the single-dose study
1 time point: sampling at 6 h after the last dose in the repeated-dose study

Colcemide (4 mg/kg bw i.p) was given 2 h prior to kill.

Duration of treatment / exposure:

single administration ("acute") and repeated administration (5 times, "subacute")

Frequency of treatment:

1x, and 5x(1x/d)

Remarks:

Doses / Concentrations:
acute and subacute: 15, 150, 1500 mg/kg suspended in 0.85 % saline
Basis:

No. of animals per sex per dose:

Single-dose study: 5 per time point and dose = 15 per dose in total; vehicle control: 3 per time point and dose = 9 per dose in total
Repeated-dose study: 5 per dose; vehicle control: 3 per dose (report, p. 124)

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg bw

Tissues and cell types examined:

bone-marrow cells, diploids
50 metaphase spreads per animal were counted (=> 250 in total per dose and pos. control; 150 in total in the control ).
Mitotic indices based at least on 500 cells: ratio of cells in mitosis/total cell count

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: pre-test on toxicity

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: Standard fixation and staining with Giemsa. Duplicate slides were prepared.

METHOD OF ANALYSIS: Light microscopy, only diploid cells analysed and scored for chromosomal aberrations

Statistics:

not required

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Metaphase aberrations: single dose (from Report Table p. 73)

Silene Dosage [mg/kg bw]	Time [h]	No. of cells	Mitotic index^1)	% cells with breaks	% cells with reunion	% cells with other aberr.^2)	% cells aberr.
15	6	250	7	0	0	0	0
	24	250	8	1	0	0	1
	48	250	12	2	0	0	2
150	6	250	7	1	0	0	1
	24	250	8	0	0	0	0
	48	250	12	3	0	0	3
1500	6	250	14	0	0	0	0
	24	250	14	3	0	0	3
	48	250	10	2	0	0	2
Saline	6	150	11	0	0	0	0
	24	150	9	3	0	0	3
	48	150	14	2	0	0	2
TEM (0.3)	48	250	4	22	12	6(a)	40

¹⁾% cells in mitosis: 500 cells observed/animal

²⁾Cells with polyploidy, pulverisation (pp), or greater than 10 aberrations (a)

Metaphase aberrations: repeated dose (5x, 1x/d) (from Report Table p. 74)

Silene Dosage [mg/kg bw]	Time after last dose [h]	No. of cells	Mitotic index^1)	% cells with breaks	% cells with reunion	% cells with other aberr.^2)	% cells aberr.
15	6	250	8	0	0	0	0
150	6	250	8	3	0	0	3
1500	6	250	7	0	0	0	0
Saline	6	150	10	2	0	0	2

¹⁾% cells in mitosis: 500 cells observed/animal

²⁾Cells with polyploidy, pulverisation (pp), or greater than 10 aberrations (a)

Conclusions:

Negative

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The use of data derived for calcium oxosilanediolate are justified for read-across to synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for calcium oxosilanediolate and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline available

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Principles of method if other than guideline:

Analysis of metaphase-chromosomes from isolated bone-marrow cells after colcemid-induced arrest of cell division in the metaphase

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: 10 - 12 weeks

Route of administration:

oral: gavage

Vehicle:

- vehicle: 0.85 % saline
- Volume of the vehicle: no data
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data


MAXIMUM DOSE VOLUME APPLIED: no data

Details on exposure:

3 time points: sampling at 6, 24, and 48 h in the single-dose study
1 time point: sampling at 6 h after the last dose in the repeated-dose study

Colcemide (4 mg/kg bw i.p) was given 2 h prior to kill.

Duration of treatment / exposure:

single administration ("acute") and repeated administration (5 times, "subacute")

Frequency of treatment:

1x, and 5x(1x/d)

Remarks:

Doses / Concentrations:
acute and subacute: 15, 150, 1500 mg/kg suspended in 0.85 % saline
Basis:

No. of animals per sex per dose:

Single-dose study: 5 per time point and dose = 15 per dose in total; vehicle control: 3 per time point and dose = 9 per dose in total
Repeated-dose study: 5 per dose; vehicle control: 3 per dose (report, p. 124)

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg bw

Tissues and cell types examined:

bone-marrow cells, diploids
50 metaphase spreads per animal were counted (=> 250 in total per dose and pos. control; 150 in total in the control ).
Mitotic indices based at least on 500 cells: ratio of cells in mitosis/total cell count

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: pre-test on toxicity

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: Standard fixation and staining with Giemsa. Duplicate slides were prepared.

METHOD OF ANALYSIS: Light microscopy, only diploid cells analysed and scored for chromosomal aberrations

Statistics:

not required

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Metaphase aberrations: single dose (from Report Table p. 73)

Silene Dosage [mg/kg bw]	Time [h]	No. of cells	Mitotic index^1)	% cells with breaks	% cells with reunion	% cells with other aberr.^2)	% cells aberr.
15	6	250	7	0	0	0	0
	24	250	8	1	0	0	1
	48	250	12	2	0	0	2
150	6	250	7	1	0	0	1
	24	250	8	0	0	0	0
	48	250	12	3	0	0	3
1500	6	250	14	0	0	0	0
	24	250	14	3	0	0	3
	48	250	10	2	0	0	2
Saline	6	150	11	0	0	0	0
	24	150	9	3	0	0	3
	48	150	14	2	0	0	2
TEM (0.3)	48	250	4	22	12	6(a)	40

¹⁾% cells in mitosis: 500 cells observed/animal

²⁾Cells with polyploidy, pulverisation (pp), or greater than 10 aberrations (a)

Metaphase aberrations: repeated dose (5x, 1x/d) (from Report Table p. 74)

Silene Dosage [mg/kg bw]	Time after last dose [h]	No. of cells	Mitotic index^1)	% cells with breaks	% cells with reunion	% cells with other aberr.^2)	% cells aberr.
15	6	250	8	0	0	0	0
150	6	250	8	3	0	0	3
1500	6	250	7	0	0	0	0
Saline	6	150	10	2	0	0	2

¹⁾% cells in mitosis: 500 cells observed/animal

²⁾Cells with polyploidy, pulverisation (pp), or greater than 10 aberrations (a)

Conclusions:

Negative

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline available

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Principles of method if other than guideline:

Fertility index, pre-implantation loss and lethal effects on embryos were determined and compared with the same parameters calculated from negative and positive control animals.

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: 10 - 12 weeks

Route of administration:

oral: gavage

Vehicle:

- vehicle: 0.85 % saline
- Volume of the vehicle: no data
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data


MAXIMUM DOSE VOLUME APPLIED: no data

Duration of treatment / exposure:

single administration ("acute") and repeated administration (5 times, "subacute")

Frequency of treatment:

1x and 5x (1x/day)

Remarks:

Doses / Concentrations:
150, 500 and 1500 mg/kg bw, suspended in 0.85 % saline, administered 1x/d (Test I), 5000 mg/kg bw (Test II)
Basis:

No. of animals per sex per dose:

10 males (treated) mated to virgin female rats (2 females/1 male)

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

triethylene melamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg bw

Tissues and cell types examined:

Fertility index = No. pregnant females / No. mated
Total No. of implantations
Total number of corpora lutea
Preimplantation losses
see Report p. 132 - 135:
Dead implants
Females with one or more dead implants
Dead implants per total implants

Statistics:

Chi-square test, Armitage´s trend test, regression analyses, Freeman-Tukey transformation, t-test (Report p. 132 - 135)

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

For single and repeated dosage of 15, 15, and 150 mg/kg bw (Report p. 80 - 99) as well as single and repeated dosage

of 5000 mg/kg bw (Report p. 100 - 117), there was no dose-response and time-trend pattern of effects

following the silicate treatment: The values calculated for reproduction parameters (see "Examinations" above)

that related to the treated animals did not significantly vary from those obtained from the negative controls,

whereas TEM caused a significant preimplantation loss and embryo resorption during the first five weeks.

Conclusions:

Non-mutagenic

Reference 4

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The use of data derived for calcium oxosilanediolate are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for calcium oxosilanediolate and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline available

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Principles of method if other than guideline:

Fertility index, pre-implantation loss and lethal effects on embryos were determined and compared with the same parameters calculated from negative and positive control animals.

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: 10 - 12 weeks

Route of administration:

oral: gavage

Vehicle:

- vehicle: 0.85 % saline
- Volume of the vehicle: no data
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data


MAXIMUM DOSE VOLUME APPLIED: no data

Duration of treatment / exposure:

single administration ("acute") and repeated administration (5 times, "subacute")

Frequency of treatment:

1x and 5x (1x/day)

Remarks:

Doses / Concentrations:
150, 500 and 1500 mg/kg bw, suspended in 0.85 % saline, administered 1x/d (Test I), 5000 mg/kg bw (Test II)
Basis:

No. of animals per sex per dose:

10 males (treated) mated to virgin female rats (2 females/1 male)

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

triethylene melamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg bw

Tissues and cell types examined:

Fertility index = No. pregnant females / No. mated
Total No. of implantations
Total number of corpora lutea
Preimplantation losses
see Report p. 132 - 135:
Dead implants
Females with one or more dead implants
Dead implants per total implants

Statistics:

Chi-square test, Armitage´s trend test, regression analyses, Freeman-Tukey transformation, t-test (Report p. 132 - 135)

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

For single and repeated dosage of 15, 15, and 150 mg/kg bw (Report p. 80 - 99) as well as single and repeated dosage

of 5000 mg/kg bw (Report p. 100 - 117), there was no dose-response and time-trend pattern of effects

following the silicate treatment: The values calculated for reproduction parameters (see "Examinations" above)

that related to the treated animals did not significantly vary from those obtained from the negative controls,

whereas TEM caused a significant preimplantation loss and embryo resorption during the first five weeks.

Conclusions:

Non-mutagenic

Reference 5

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

The host organism is inoculated by intraperitoneal injection with a common indicator microorganism/tester strain before treatment with the test substance. After "incubation" in the host organism, the tester strain is withdrawn from the ascites and tested for mutation on minimal agar plate., e.g. according to Ames.

GLP compliance:

no

Type of assay:

other: Host mediated assay

Species:

mouse

Strain:

ICR

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

single administration ("acute") and repeated administration (5 times, "subacute")

Frequency of treatment:

1x and 5x (1x/d)

Remarks:

Doses / Concentrations:
acute and subacute: 15, 150, 1500 and 5000 mg/kg suspended in 0.85 % saline
Basis:

Positive control(s):

Dimethylnitrosamine (DMN)

Key result

Sex:

male

Genotoxicity:

negative

Positive controls validity:

valid

There was a high increase in mutants following oral treatment with DMN, 

but no significant increases in mutation rates at any dose and dose regimen.

Conclusions:

Negative

Reference 6

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The use of data derived for calcium oxosilanediolate are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for calcium oxosilanediolate and synthetic wollastonite. Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline available

Principles of method if other than guideline:

The host organism is inoculated by intraperitoneal injection with a common indicator microorganism/tester strain before treatment with the test substance. After "incubation" in the host organism, the tester strain is withdrawn from the ascites and tested for mutation on minimal agar plate., e.g. according to Ames.

GLP compliance:

no

Type of assay:

other: Host mediated assay

Species:

mouse

Strain:

ICR

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

single administration ("acute") and repeated administration (5 times, "subacute")

Frequency of treatment:

1x and 5x (1x/d)

Remarks:

Doses / Concentrations:
acute and subacute: 15, 150, 1500 and 5000 mg/kg suspended in 0.85 % saline
Basis:

Positive control(s):

Dimethylnitrosamine (DMN)

Key result

Sex:

male

Genotoxicity:

negative

Positive controls validity:

valid

There was a high increase in mutants following oral treatment with DMN, 

but no significant increases in mutation rates at any dose and dose regimen.

Conclusions:

Negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro gene mutation study in bacteria (Ames tests):

In the key study (Brown 2021) Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^-were treated with suspensions of the test material Synthetic wollastonite using both the Ames plate incorporation and pre-incubation methods at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 1.5 to 5000 µg/plate in the first experiment.The experiment was repeated on a separate day (pre-incubation method) using the dose range 15 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test material formulations. 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. The test material was therefore considered to be non-mutagenic under the conditions of the test.

In the supporting study (Thompson 2010) Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^-were treated with suspensions of the test material Kieselguhr soda ash flux calcined using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment.The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.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. The test material was therefore considered to be non-mutagenic under the conditions of the test.

Read-Across Justification for genetic toxicity

Synthetic wollastonite is a UVCB substance, the main constituents of which are amorphous in nature. It also contains crystalline silica. The percentage of crystalline silica may range up to 1.5% (<=0.21% respirable). The genetic toxicity information has been read-across from the analogue substance Kieselguhr, soda ash flux-calcined for the in-vitro studies and from Silicic acid, calcium salt for the in-vivo studies. The analogues have been chosen for their similarity in structure and properties to Synthetic wollastonite. The main difference in structure between Synthetic wollastonite and Kieselguhr, soda ash flux-calcined is the presence of a calcium ion in Synthetic wollastonite. It is clear from a number of studies carried out on amorphous and crystalline silica and the analogue substance Silicic acid, calcium salt that results were consistent between the two substances and that there was no effect from the presence of the calcium ion. Moreover, it is well documented that these substances have a low potential for hazard to health and the environment. 

The toxicological properties both forms are well described and may be used to predict the genotoxicity effects of exposure to Synthetic wollastonite , and to support the available data for this substance.

In vitro cytogenicity study in mammalian cells (chromosome aberration test):

In the key study (Morris 2010) duplicate cultures of human lymphocytes, treated with the read-across test material Kieselguhr soda ash flux calcined, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24 hours continuous exposure in the absence of metabolic activation.

The test material was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included a dose level that was the lowest precipitating dose level.

 

In vitro gene mutation study in mammalian cells (mouse lymphoma assay):

In the key study (Flanders 2010) carried out on the read-across test material Kieselguhr soda ash flux calcined, one main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels in duplicate, together with vehicle (R0 medium) and positive controls. The exposure groups used were as follows: 4-hour exposures both with and without metabolic activation, and 24–hour exposure without metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity and was 2.5 to 40 μg/ml for all three of the exposure groups. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

 

In vivo: Genetic studies:

Three studies carried out on the read-across substance calcium oxosilanediolate (silicic acid, calcium salt) concluded that the substance was negative for cytogenicity and mutagenicity.

 

Short description of key information:

Synthetic wollastonite was tested for genotoxicity in an Ames (OECD TG 471). The read-across test material Kieselguhr soda ash flux calcined was tested in in vitro studies for genotoxicity in an Ames (OECD TG 471) chromosome aberration (OECD TG 473) and a mouse lymphoma assay (OECD TG 476). Results were negative in all three tests.  

A further three in vivo studies carried out on the read-across material calcium oxosilanediolate concluded that the read-across substance should not be considered to be cytotoxic or mutagenic.

Justification for classification or non-classification

Results were negative in a Ames study carried out on Synthetic wollastonite. Negative results were also obtained from a set of reliable in vitro genotoxicity studies carried out on the read-across material Kieselghur soda ash flux calcined therefore no classification is warranted.

Results were also negative from three in vivo studies carried out on the read-across material calcium oxosilanediolate.

Based on the results of testing on the analogue substance Kieselghur soda ash flux calcined, and the analogue substance calcium oxosilanediolate, the test substance Synthetic Wollastonite can be considered to be not classified for genotoxicity (cytogenicity and mutagenicity).