Cordierite (Mg2[Al4O3(SiO3)5])

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro gene mutation study in bacteria was conducted on the registered substance according to OECD Testing Guideline 471. The test item was considered to be non-mutagenic in bacteria under the conditions of the test, for both methods, with and without metabolic activation.

An assessment of the capacity of the registered substance to induce gene mutation in mammalian cells was conducted based on an examination of the composition of the substance and the potential of its constituents to induce gene mutation in mammalian cells. This assessment was supported by the toxicokinetics profile of the substance and data from a similar substance. It was concluded that the registered substance is not expected to induce gene mutation in mammalian cells. The registered substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The in vitro cytogenicity study was conducted on a read-across substance according to a method similar to OECD Testing Guideline 473. The test item was considered to be non-mutagenic in mammalian cells under the conditions of the test.

The in vivo cytogenicity study was conducted on a read-across substance according to a method similar to OECD Testing Guideline 475. The test item was considered to be non-mutagenic in vivo under the conditions of the test.

Therefore, the registered substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 15 July 2015 to 24 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 471. The study was conducted on the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

TA1537: his C 3076; rfa-; uvrB-
TA98: his D 3052; rfa-; uvrB-;R-factor
TA1535: his G 46; rfa-; uvrB-
TA100: his G 46; rfa-; uvrB-;R-factor
WP2uvrA: trp-; uvrA-

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method
The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- 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. In situations where the test item is insoluble with the standard range of Ames vehicles, the solvent which produces the best doseable suspension is sought. The test item formed the best doseable suspension in dimethyl sulphoxide; therefore, this solvent was selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

1) Test for Mutagenicity (Experiment 1) – Plate Incorporation Method

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described without metabolic activation except that 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.

Incubation and Scoring
All of the plates were incubated at 37°C ± 3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to revertant colonies spreading slightly, thus distorting the actual plate count.

2) Test for Mutagenicity (Experiment 2) – Pre-Incubation Method

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37°C ± 3°C for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously (see 3.5.2.2) except that 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°C ± 3°C for 20 minutes (with shaking) and addition of molten amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37°C ± 3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to revertant colonies spreading slightly (and one artefact on the plate), thus distorting the actual plate count.

3) Acceptance 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).
plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
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. The historical ranges of the positive control reference items for 2013 and 2014 are presented in Appendix 1.
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. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test item precipitate (powdery in appearance after the plate incorporation method but cloudy after employing the pre-incubation modification) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
There were no significant 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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9-mix at 15 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 15 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.
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'.

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
75 64 68	(69)	25 29 24	(26)	24 16 24	(21)	29 17 17	(21)	8 8 9	(8)

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type				Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
114 124 124	(121)	19 21 17	(19)	33 21 25	(26)	21 16 7	(15)	6 17 10	(11)

Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 16 July 2015					To: 19 July 2015
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)	78 74 76	(76) 2.0#	23 27 29	(26) 3.1	11 21 11	(24) 5.8	15 12 19	(15) 3.5	16 8 12	(12) 4.0
	1.5 µg	65 61 68	(65) 3.5	32 21 28	(27) 5.6	23 13 15	(17) 5.3	8 32 21	(20) 12.0	16 16 11	(14) 2.9
	5 µg	60 78 67	(68) 9.1	25 25 21	(24) 2.3	16 23 9	(16) 7.0	11 21 13	(15) 5.3	13 16 9	(13) 3.5
	15 µg	80 71 75	(75) 4.5	24 26 31	(27) 3.6	15 20 20	(18) 2.9	19 15 20	(18) 2.6	13 15 12	(13) 1.5
	50 µg	83 79 68	(77) 7.8	28 27 21	(25) 3.8	13 17 13	(14) 2.3	11 19 12	(14) 4.4	17 9 23	(16) 7.0
	150 µg	61 69 60	(63) 4.9	31 28 32	(30) 2.1	20 19 13	(17) 3.8	12 16 17	(15) 2.6	5 15 16	(12) 6.1
	500 µg	64 74 75	(71) 6.1	28 30 29	(29) 1.0	19 15 13	(16) 3.1	12 12 9	(11) 1.7	12 11 11	(11) 0.6
	1500 µg	79 P 63 P 76 P	(73) 8.5	25 P 27 P 30 P	(27) 2.5	12 P 11 P 21 P	(15) 5.5	9 P 17 P 17 P	(14) 4.6	13 P 9 P 16 P	(13) 3.5
	5000 µg	67 P 80 P 60 P	(69) 10.1	30 P 28 P 15 P	(29) 1.0	13 P 23 P 15 P	(17) 5.3	23 P 23 P 20 P	(22) 1.7	15 P 9 P 7 P	(10) 4.2
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		368 369 445	(394) 44.2	508 631 568	(569) 61.5	639 555 533	(576) 55.9	171 206 179	(185) 18.3	1236 974 730	(980) 253.1

Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 16 July 2015					To: 19 July 2015
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)	61 64 78	(68) 9.1#	23 26 30	(26) 3.5	25 13 31	(23) 9.2	17 24 24	(22) 4.0	9 13 16	(13) 3.5
	1.5 μg	69 82 78	(76) 6.7	27 21 29	(26) 4.2	28 25 11	(21) 9.1	16 20 16	(17) 2.3	8 15 19	(14) 5.6
	5 μg	86 69 79	(78) 8.5	28 24 28	(27) 2.3	27 17 32	(25) 7.6	19 20 19	(19) 0.6	16 12 13	(14) 2.1
	15 μg	76 79 61	(72) 9.6	30 27 26	(28) 2.1	8 28 20	(19) 10.1	29 16 15	(20) 7.8	17 12 17	(15) 2.9
	50 μg	72 74 72	(73) 1.2	21 25 26	(24) 2.6	23 19 15	(19) 4.0	19 12 24	(18) 6.0	13 12 17	(14) 2.6
	150 μg	76 78 72	(75) 3.1	26 29 28	(28) 1.5	32 28 17	(26) 7.8	21 25 27	(24) 3.1	4 10 9	(8) 3.2
	500 μg	68 69 65	(67) 2.1	27 30 24	(27) 3.0	24 13 15	(17) 5.9	17 20 12	(16) 4.0	16 9 12	(12) 3.5
	1500 μg	79 P 63 P 65 P	(69) 8.7	23 P 32 P 24 P	(26) 4.9	21 P 21 P 20 P	(21) 0.6	17 P 9 P 19 P	(15) 5.3	16 P 12 P 13 P	(14) 2.1
	5000 μg	68 P 74 P 86 P	(76) 9.2	29 P 24 P 23 P	(25) 3.2	20 P 16 P 19 P	(18) 2.1	8 P 19 P 29 P	(19) 10.5	9 P 16 P 18 P	(14) 4.7
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 μg		2 μg		10 µg		5 µg		2 µg
		772 905 938	(872) 87.9	254 277 259	(263) 12.1	172 212 191	(192) 20.0	131 171 140	(147) 21.0	382 345 303	(343) 39.5

Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 21 July 2015					To: 24 July 2015
S9-Mix(-)	Dose LevelPer Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control(DMSO)	879883	(89)7.8#	16917	(14)4.4	231719	(20)3.1	122325	(20)7.0	7127	(9)2.9
	15 μg	837272	(76)6.4	201723	(20)3.0	202011	(17)5.2	282119	(23)4.7	6126	(8)3.5
	50 μg	796980	(76)6.1	191729	(22)6.4	212723	(24)3.1	15829	(17)10.7	11107	(9)2.1
	150 μg	9110780	(93)13.6	241210	(15)7.6	252525	(25)0.0	171319	(16)3.1	13149	(12)2.6
	500 μg	696676	(70)5.1	211311	(15)5.3	251625	(22)5.2	161524	(18)4.9	14117	(11)3.5
	1500 μg	94 P87 P75 P	(85)9.6	13 P12 P13 P	(13)0.6	25 P21 P24 P	(23)2.1	33 P13 P20 P	(22)10.1	8 P9 P9 P	(9)0.6
	5000 μg	68 P87 P98 P	(84)15.2	22 P15 P17 P	(18)3.6	25 P20 P23 P	(23)2.5	9 P15 P13 P	(12)3.1	10 P13 P13 P	(12)1.7
PositivecontrolsS9-Mix(-)	NameDose LevelNo. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 μg		5 μg		2 μg		0.2 μg		80 μg
		707581730	(673)80.2	657711806	(725)75.4	9681085976	(1010)65.4	329343346	(339)9.1	6991033684	(805)197.3

Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 21 July 2015					To: 24 July 2015
S9-Mix(+)	Dose LevelPer Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control(DMSO)	1119295	(99)10.2#	172121	(20)2.3	434129	(38)7.6	232233	(26)6.1	131911	(14)4.2
	15 μg	112128139	*(126)13.6	9128	(10)2.1	443123	(33)10.6	133224	(23)9.5	61112	(10)3.2
	50 μg	102104111	(106)4.7	111014	(12)2.1	272125	(24)3.1	132913	(18)9.2	12715	(11)4.0
	150 μg	11498110	(107)8.3	1297	(9)2.5	312725	(28)3.1	182117	(19)2.1	121210	(11)1.2
	500 μg	11991111	(107)14.4	131615	(9)2.6	243228	(28)4.0	261921	(22)3.6	14914	(12)2.9
	1500 μg	118 P107 P100 P	(108)9.1	7 P8 P12 P	(9)1.0	31 P17 P32 P	(27)8.4	15 P20 P17 P	(17)2.5	8 P12 P11 P	(10)2.1
	5000 μg	103 P95 P90 P	(96)6.6	8 P10 P9 P	(9)1.0	32 P23 P25 P	(27)4.7	14 P13 P15 P	(14)1.0	11 P8 P9 P	(9)1.5
PositivecontrolsS9-Mix(+)	NameDose LevelNo. of Revertants	2AA		2AA		2AA		BP		2AA
		1 μg		2 μg		10 μg		5 μg		2 μg
		525663629	(606)71.9	199234250	(228)26.1	285277243	(268)22.3	119144146	(136)15.0	294287279	(287)7.5

2AA 2-Aminoanthracene

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

BP Benzo(a)pyrene

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

P Test item precipitate

* p≤0.05

# Standard deviation

Conclusions:

Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic under the conditions of the test, for both methods, with and without metabolic activation.

Executive summary:

The in vitro genotoxicity in bacteria of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 471. Ames plate incorporation (Experiment 1) and pre-incubation (Experiment 2) methods were performed at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

There were no significant 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 metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9-mix at 15 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 15 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control. The test item was therefore considered to be non-mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: Expert assessment

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

An assessment was conducted based on an examination of the composition of the substance and the potential of its constituents to induce gene mutation in mammalian cells. This assessment was supported by the toxicokinetics profile of the substance and data from a similar substance.

Qualifier:

no guideline required

Principles of method if other than guideline:

An assessment was conducted based on an examination of the composition of the substance and the potential of its constituents to induce gene mutation in mammalian cells. This assessment was supported by the toxicokinetics profile of the substance and data from a similar substance.

GLP compliance:

no

Type of assay:

other: An assessment was conducted based on an examination of the composition of the substance and the potential of its constituents to induce chromosomal aberration in mammalian cells, supported by data from a similar substance and toxicokinetics behaviour.

Genotoxicity:

negative

Remarks on result:

other: An assessment was performed based on the toxicokinetic behaviour on the substance and QSAR predictions.

An assessment was conducted based on an examination of the composition of the substance and the potential of its constituents to induce gene mutation in mammalian cells. This assessment was supported by the toxicokinetics profile of the substance and data from a similar substance.

Conclusions:

Interpretation of results (migrated information):
negative

Based on an examination of the toxicokinetics profile of the substance, the capacity of its constituent to induce gene mutation, and data from a similar substance, it is concluded that the registered substance is not expected to induce gene mutation in mammalian cells.

Executive summary:

The potential of the registered substance to induce gene mutation in mammalian cells has been assessed based on the toxicokinetic behaviour of the substance and the results obtained on the analogous substance SAS (Johnston et al., 2000). Based on the toxicokinetic behaviour of the registered substance, the substance is not expected to be absorbed and negligible release of the constituents of the registered substance would occur in the GI tract following an oral exposure. These constituents are expected to be poorly absorbed, leading to a systemic exposure considered as inconsequential when compared to normal dietary exposure, and therefore will not induce detectable genotoxic effects. The analogous substance SAS was assessed for its capacity to induce gene mutation in mammalian cells and provided negative results. It is concluded that the registered substance is not expected to induce gene mutation in mammalian cells. On the basis of these results an experimental study on the potential of the registered substance to induce gene mutation in mammalian cells can be avoided since it is unlikely to provide additional relevant data about the in vivo mutagenicity potential of the registered substance in accordance with ECHA’s Endpoint Specific Guidance on Genotoxicity.

Johnston C, Driscoll K, Finkelstein J, Baggs R, O'Reilly M, Carter J, Gelein R, Oberdörster G (2000) Pulmonary chemokine and mutagenic responses in rats after subchronic inhalation of amorphous and crystalline silica. Toxicol Sci. 2000 Aug;56(2):405-13.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted using a method similar to OECD Testing Guideline 473 and meets acceptable scientific standards.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Study performed without metabolic activation using a non-standard cell lines (WI-38 - human embryonic lung cultures)

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: WI-38 - human embryonic lung cultures

Details on mammalian cell type (if applicable):

- Type and identity of media: Not provided
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: Not provided
- Periodically "cleansed" against high spontaneous background: Not provided

Metabolic activation:

without

Test concentrations with justification for top dose:

1, 10 and 100 µg/ml

Vehicle / solvent:

- Vehicle: 0.85 saline

Negative solvent / vehicle controls:

yes

Remarks:

Saline solution

Positive controls:

yes

Positive control substance:

triethylenemelamine

Remarks:

0.1 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 24 - 48 h

STAIN: acetic acid-orcein stain (20%)

NUMBER OF REPLICATIONS: 3 per concentration

NUMBER OF CELLS EVALUATED: 100 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy

Evaluation criteria:

Evaluation of the genetic damage by identification of the aberrations (bridges, pseudochiasmata, multipolar cells, acentric fragments, etc) induced by the test substance.

Statistics:

Not provided

Species / strain:

mammalian cell line, other: WI-38 - human embryonic lung cultures

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

250 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Acentric fragments:
- Negative control: 2%
- 1 µg/ml: 0%
- 10 µg/ml: 1%
- 100 µg/ml: one cell with an acentric fragment and one cell with a bridge (non significant).
- Positive control: four cells with pulverization together with the other aberrations.

Remarks on result:

other: strain/cell type: WI-38 - human embryonic lung cultures

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

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

Executive summary:

The in vitro cytogenicity in mammalian cells of the read-across substance sodium silicoaluminate branded as FDA 71-45 was determined according to a method similar to the OECD Guideline for Testing of chemicals 473. Chromosome aberrations were evaluated in WI-38 - human embryonic lung cultures without metabolic evaluation.

The in vitro mammalian chromosome aberration test was performed with concentrations of 1, 10 and 100 µg/ml in a saline solution at 0.85%. Anaphase preparations were made by growing the WI-38 cells in presence of the test item. Cells were then suspended in acetic acid-orcein stain (20%) and put in slides to be evaluated. Evaluation of the genetic damage was made by identification of the aberrations induced by the test item.

Cytotoxicity was observed at a concentration of 250 µg/ml. No significant numbers of aberration were observed for each tested concentration.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

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

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted using a method similar to OECD Testing Guideline 475 and meets acceptable scientific standards.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

higher dose limit, 500 cells evaluated per animal instead of 1000

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: Not provided
- Age at study initiation: 10 to 12 weeks
- Weight at study initiation: 280 to 350 g
- Assigned to test groups randomly: Yes
- Fasting period before study: Not provided
- Housing: Five per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Quarantine for 4 - 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not provided
- Humidity (%): Not provided
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): Not provided

Route of administration:

oral: gavage

Vehicle:

- Vehicle: 0.85 saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Not provided
- Mixing appropriate amounts with (Type of food): Not provided
- Storage temperature of food: Not provided

Duration of treatment / exposure:

Acute toxicity: single administration
Subacute toxicity: five days (five administrations)

Frequency of treatment:

Acute toxicity: one exposure at three time points (6 hours, 24 hours, 48 hours)
Subacute toxicity: one exposure per concentration per day

Post exposure period:

Termination after administration

Remarks:

Doses / Concentrations:
subacute: 4.25, 42.5, 425 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
acute: 5000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

Acute toxicity: five per dose per time points (45) + negative control three per time points (9) + positive control five only for the last time point (5)
Subacute toxicity: five per dose (15) + negative control

Control animals:

yes, concurrent vehicle

other: positive control: TEM 0.3 mg/kg

Positive control(s):

triethylenemelamine (TEM)
- Route of administration: oral gavage
- Doses / concentrations: 0.3 mg/kg bw

Tissues and cell types examined:

Bone marrow diploid cells
Fifty metaphase spreads scored per animal.
Mitotic indices obtained by counting at least 500 cells

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
- Slides dried at room temperature. Duplicate slides prepared. Slides stained using a 5% Giemsa solution (Giemsa buffer pH 7.2) for 20 minutes, rinsed in acetone, 1:1 acetone:xylene, and placed in fresh xylene for 30 minutes.

METHOD OF ANALYSIS:
- Chromosomes of each cell counted and only diploid cells analyzed.

OTHER:
- Intraperitoneal administration of 4 mg/kg of colcemid two hours prior to killing.

Evaluation criteria:

Chromatid gaps and breaks, polyploidy, chromosome gaps and breaks, reunions, pulverization, cells with greater than ten aberrations, and any other chromosomal aberrations.

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information): negative
The test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

The in vivo cytogenicity of the read-across substance sodium silicoaluminate branded as FDA 71-45 was determined according to a method similar to the OECD Guideline for Testing of chemicals 475. Chromosome aberrations were evaluated in Sprague-Dawley rats.

Two methods of dosing were used. In the acute method, one dose of 5000 mg/kg bw was administrated. In the subacute, doses of 4.25, 42.5, and 425 mg/kg were administrated daily for five days.

Each animal was intraperitoneally administrated 4 mg/kg of colcemid two hours prior to killing in order to stop the mitosis. Bone marrow slides were prepared and stained with 5% Giemsa solution in order to score pulverization, achromatid gaps and breaks, chromosome gaps and breaks, cells with greater than ten aberrations, reunions, polyploidy, and any other chromosomal aberrations in diploid cells.

No significant numbers of aberration were observed for each tested concentration.

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

Additional information

Additional information from genetic toxicity in vitro:

The in vitro genotoxicity in bacteria of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 471. Ames plate incorporation (Experiment 1) and pre-incubation (Experiment 2) methods were performed at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). There were no significant 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 metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9-mix at 15 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 15 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control. The test item was therefore considered to be non-mutagenic.

The potential of the registered substance to induce gene mutation in mammalian cells has been assessed based on the toxicokinetic behaviour of the substance and the results obtained on the analogous substance synthetic amorphous silica (SAS). Based on the toxicokinetic behaviour of the registered substance, the substance is not expected to be absorbed and negligible release of the constituents of the registered substance would occur in the GI tract following an oral exposure. These constituents are expected to be poorly absorbed, leading to a systemic exposure considered as inconsequential when compared to normal dietary exposure, and therefore will not induce detectable genotoxic effects. The analogous substance SAS was assessed for its capacity to induce gene mutation in mammalian cells and provided negative results. It is concluded that the registered substance is not expected to induce gene mutation in mammalian cells. On the basis of these results an experimental study on the potential of the registered substance to induce gene mutation in mammalian cells can be avoided since it is unlikely to provide additional relevant data about the in vivo mutagenicity potential of the registered substance in accordance with ECHA’s Endpoint Specific Guidance on Genotoxicity.

The in vitro cytogenicity in mammalian cells of the read-across substance sodium silicoaluminate branded as FDA 71-45 was determined according to a method similar to the OECD Guideline for Testing of chemicals 473. Chromosome aberrations were evaluated in WI-38 - human embryonic lung cultures without metabolic evaluation. The in vitro mammalian chromosome aberration test was performed with concentrations of 1, 10 and 100 µg/ml in a saline solution at 0.85%. Anaphase preparations were made by growing the WI-38 cells in presence of the test item. Cells were then suspended in acetic acid-orcein stain (20%) and put in slides to be evaluated. Evaluation of the genetic damage was made by identification of the aberrations induced by the test item. Cytotoxicity was observed at a concentration of 250 µg/ml. No significant numbers of aberration were observed for each tested concentration. The test item was considered to be non-mutagenic under the conditions of the test.

The in vivo cytogenicity of the read-across substance sodium silicoaluminate branded as FDA 71-45 was determined according to a method similar to the OECD Guideline for Testing of chemicals 475. Chromosome aberrations were evaluated in Sprague-Dawley rats. Two methods of dosing were used. In the acute method, one dose of 5000 mg/kg bw was administrated. In the subacute, doses of 4.25, 42.5, and 425 mg/kg were administrated daily for five days. Bone marrow slides were prepared and stained with 5% Giemsa solution in order to score pulverization, achromatid gaps and breaks, chromosome gaps and breaks, cells with greater than ten aberrations, reunions, polyploidy, and any other chromosomal aberrations in diploid cells. No significant numbers of aberration were observed for each tested concentration. The test item was considered to be non-mutagenic under the conditions of the test.

Justification for selection of genetic toxicity endpoint

This study was selected as the key study as it was a GLP study conducted on the registered substance according to OECD Testing Guideline 471. The results of this study are considered as representative for this endpoint.

Justification for classification or non-classification

The in vitro gene mutation study in bacteria was conducted on the registered substance according to OECD Testing Guideline 471. The test item was considered to be non-mutagenic in bacteria under the conditions of the test, for both methods, with and without metabolic activation.

An assessment of the capacity of the registered substance to induce gene mutation in mammalian cells was conducted based on an examination of the composition of the substance and the potential of its constituents to induce gene mutation in mammalian cells. This assessment was supported by the toxicokinetics profile of the substance and data from a similar substance. It was concluded that the registered substance is not expected to induce gene mutation in mammalian cells.

The in vitro cytogenicity study was conducted on a read-across substance according to a method similar to OECD Testing Guideline 473. The test item was considered to be non-mutagenic in mammalian cells under the conditions of the test.

The in vivo cytogenicity study was conducted on a read-across substance according to a method similar to OECD Testing Guideline 475. The test item was considered to be non-mutagenic in vivo under the conditions of the test.

Therefore, the registered substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.