Aluminum chloride, basic, reaction products with silica

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered (fibrous) substance was shown not to be mutagenic in a bacterial gene mutation (Ames) test or in a human cell chromosome damage study. Further in vitro testing (e.g. in a mammalian cell gene mutation assay) was considered but concluded to be scientifically unnecessary and unwarranted in view of the chemistry and physical form of the substance.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Simple protocol study except that a particulate was tested causing the appearance of a precipitate on the slides. No studies were carried out on the homogeneity of the test item or its solubility, however it did not effect pH or osmolality of culture medium at concentrations up to 1mg/ml.

Qualifier:

according to guideline

Guideline:

other: Draft OECD 487

Deviations:

yes

Remarks:

Test article was particulate inorganic solid

Principles of method if other than guideline:

Cultures of human lymphocytes evaluated for the presence of micronuclei in binucleate cells at 3 dose levels.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

None

Species / strain / cell type:

lymphocytes:

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

31.88 - 1020 mg/ml

Vehicle / solvent:

Minimum essential medium (MEM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: & Cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 & 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours & 32 hours


NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration of test substance

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis block proliferation index

Evaluation criteria:

Criteria for identifying micronuclei was that they were round or oval, non refractile not linked to the main nucleus and were less than a third of the diameter of the main nucleus.

Statistics:

The frequency of cells with micronuclei was compared with the concurrent control value using a chi squared test. A significant result was recorded if the p value was less than 0.05 with a dose reponse relationship.

Species / strain:

lymphocytes:

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant effect of test article on pH
- Effects of osmolality: no significant effect of test article on osmolality
- Precipitation: Material was an insoluble inorganic solid

Remarks on result:

other: other: Micronucleus induction in human lyphocytes

Remarks:

Migrated from field 'Test system'.

Results tables for theIn vitroMN test in Human Lymphocytes on Crushed Polycrystalline Wool

Study number 3016/0003

Table 1 - CBPI - Preliminary Toxicity Test

4-hour exposure without S9							4-hour exposure with S9						20-hour exposure without S9
Dose Level (μg/ml)	Nucleate Cells/ 500 Cells			CBPI	% Control CBPI	Dose Level (μg/ml)	Nucleate Cells/ 500 Cells			CBPI	% Control CBPI	Dose Level (μg/ml)	Nucleate Cells/ 500 Cells			CBPI	% Control CBPI
	Mono	Bi	Multi				Mono	Bi	Multi				Mono	Bi	Multi
0	146	218	136	1.98	100	0	172	207	121	1.90	100	0	82	256	162	2.16	100
3.98	NS	NS	NS	NS		3.98	NS	NS	NS	NS		3.98	NS	NS	NS	NS
7.97	NS	NS	NS	NS		7.97	NS	NS	NS	NS		7.97	NS	NS	NS	NS
15.94	NS	NS	NS	NS		15.94	NS	NS	NS	NS		15.94	NS	NS	NS	NS
31.88	NS	NS	NS	NS		31.88	NS	NS	NS	NS		31.88	NS	NS	NS	NS
63.75	NS	NS	NS	NS		63.75 P	NS	NS	NS	NS		63.75	NS	NS	NS	NS
127.5 P	179	228	94	1.83	85	127.5 P	188	225	87	1.80	89	127.5 P	77	253	170	2.19	102
255 P	130	240	130	2.00	102	255 P	161	224	115	1.91	101	255 P	74	259	167	2.19	102
510 P*	155	226	119	1.93	95	510 P*	198	220	82	1.77	86	510 P*	72	269	159	2.17	101
1020 P*	167	222	111	1.89	91	1020 P*	208	194	98	1.78	87	1020 P*	86	251	163	2.15	99

NS - = Not selected for CBPI analysis

P= Undissolved solid material of the test item observed at end of exposure period in blood-free cultures

P*= Undissolved solid material of the test item observed on slides and at end of exposure period in blood-free cultures

Table 2 - CBPI and Micronucleus Data – Experiment 1 without Metabolic Activation (S9)

Dose level (μg/ml)	Exposure Time +/-S9	Replicate	Nucleate cells /500 cells			CBPI	% Control CBPI	Micronuclei (MN) Per 1000 Bi-nucleate cells			%Cells with MN	Mean % Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
0	4Hr-S9	A	230	195	75	1.69	100	4	0	0	0.40	0.45
		B	184	229	87	1.81		5	0	0	0.50
255 P		A	207	194	99	1.78	109	5	0	0	0.50	0.45
		B	166	239	95	1.86		4	0	0	0.40
510 P		A	206	197	97	1.78	107	6	0	0	0.60	0.65
		B	175	242	83	1.82		7	0	0	0.70
1020 P		A	187	214	99	1.82	111	5	0	0	0.50	0.35
		B	178	217	105	1.85		2	0	0	0.20
MMC 0.2		A	232	231	37	1.61	77	70	8	1	7.90	8.60***
		B	257	216	27	1.54		86	6	1	9.30

MMC = Mitomycin C

P= Undissolved solid material of the test item observed at end of exposure period

*** = P<0.001

Table 3 - CBPI and Micronucleus Data – Experiment 1 with Metabolic Activation (S9) (2%)

Dose level (μg/ml)	Exposure Time +/-S9	Replicate	Nucleate cells /500 cells			CBPI	% Control CBPI	Micronuclei (MN) Per 1000 Bi-nucleate cells			%Cells with MN	Mean % Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
0	4Hr+S9	A	232	193	75	1.69	100	6	0	0	0.60	0.45
		B	167	240	93	1.85		3	0	0	0.30
255P		A	205	190	105	1.8	102	6	0	0	0.60	0.55
		B	207	202	91	1.77		5	0	0	0.50
510 P		A	191	228	81	1.78	99	5	1	0	0.60	0.50
		B	202	220	78	1.75		4	0	0	0.40
1020 P		A	217	206	77	1.72	97	6	0	0	0.60	0.50
		B	198	215	87	1.77		4	0	0	0.40
CP 5		A	389	107	4	1.23	34	56	4	0	6.00	5.85***
		B	358	136	6	1.3		54	3	0	5.70

CP = Cyclophosphamide

P= Undissolved solid material of the test item observed at end of exposure period

***= P<0.001

Table 4 - CBPI and Micronucleus Data – Experiment 2 without Metabolic Activation (S9)

Dose Level (μg/ml)	Exposure Time +/-S9	Replicate	Nucleate cells / 500 cells			CBPI	% Control CBPI	Micronuclei (MN) Per 1000 Bi-nucleate cells			%Cells with MN	Mean % Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
0	20Hr-S9	A	94	228	178	2.17	100	5	0	0	0.50	0.50
		B	75	222	203	2.26		4	1	0	0.50
255 P		A	98	244	158	2.12	96	6	1	0	0.70	0.65
		B	86	221	193	2.21		5	1	0	0.60
510 P		A	114	230	156	2.08	93	5	1	0	0.60	0.35
		B	96	223	181	2.17		1	0	0	0.10
1020 P		A	84	230	186	2.20	97	6	0	0	0.60	0.30
		B	104	220	176	2.14		0	0	0	0.00
DC 0.075		A	220	215	65	1.69	57	28	6	3	3.70	4.45***
		B	219	216	65	1.69		44	3	5	5.20

DC = Demecolcine

Table 5 - CBPI and Micronucleus Data – Experiment 2 with Metabolic Activation (S9) (1%)

Dose level (μg/ml)	Exposure Time +/-S9	Replicate	Nucleate cells / 500 cells			CBPI	% Control CBPI	Micronuclei (MN) Per 1000 Bi-nucleate cells			%Cells with MN	Mean % Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
0	4Hr+S9	A	166	188	146	1.96	100	3	0	0	0.30	0.50
		B	164	210	126	1.92		6	1	0	0.70
255 P		A	166	223	111	1.89	86	6	1	0	0.70	0.55
		B	231	173	96	1.73		4	0	0	0.40
510 P		A	140	238	122	1.96	88	7	0	0	0.70	0.95
		B	235	186	79	1.69		11	1	0	1.20
1020 P		A	144	220	136	1.98	102	4	0	0	0.40	0.50
		B	169	193	138	1.94		5	0	1	0.60
CP 5		A	252	218	30	1.56	56	59	4	1	6.40	5.85***
		B	267	215	18	1.50		43	8	2	5.30

P= Undissolved solid material of the test item observed at end of exposure period

*** = P<0.001

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation & negative without metabolic activation

Polycrystalline wools are not genotoxic to human lymphocytes in vitro.

Executive summary:

In a well conducted trial with and without metabolic activation and using 2 positive control substances polycrystalline wools are not genotoxic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The registered substance is a man-made polycrystalline wool, comprising fibres with an alumina/silica composition. These fibres are essentially insoluble in water, releasing <0.01 wt% of aluminium oxide (Al2O3) and silicon dioxide (SiO2) in a 24h water extraction study. Neither aluminium oxide nor silicon dioxide is classified as a hazardous substance under EU CLP criteria, and neither has been reported to be mutagenic. Hence there is no expectation of mutagenic activity of these fibres.  Naturally occurring mineral fibres such as asbestos which (unlike the registered substance) are classified as carcinogenic have been shown to cause chromosome damage in vitro (and some have also shown evidence of DNA interaction in specific types of assay, probably associated with localised active oxygen release), but give mainly negative results in gene mutation assays (bacterial and mammalian).

The clearly negative results obtained when the registered substance was tested for micronucleus induction (chromosome damage) in cultured human lymphocytes therefore provide a firm basis for distinguishing these polycrystalline wool fibres from genotoxic mineral fibres such as asbestos. Taking account of these findings, the physical form of the fibres and their chemical composition, a conclusion of non-genotoxicity can be reached without further testing.

Justification for selection of genetic toxicity endpoint
The genotoxicity endpoints of gene mutation (in bacterial systems) and chromosome damage (in human cells) have been investigated. Chromosomal damage arising from impairment of mitosis has been recognised as a major indicator of genotoxicity for fibres showing mutagenic activity (NIOSH, 2008: Revised Draft NIOSH Current Intelligence Bulletin on Asbestos Fibers and Other Elongated Mineral Particles, State of the Science and Roadmap for Research. US Department of Health and Human Services Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, June 2008. Also Jaurand, MC, 1997: Mechanisms of Fiber-induced Genotoxicity. Environmental Health Perspectives Vol. 105 Supplement 5, 1073-1084). The endpoint investigated in the selected principal study record for genotoxicity (micronucleus test of polycrystalline wool in cultured lymphocytes) is a sensitive indicator of such chromosome damage, monitored in cultured human cells.

Justification for classification or non-classification

In vitro tests for (bacterial) gene mutation and (human cell) chromosome damage have shown no activity of the registered substance. Its chemical composition and essential water insolubility give rise to no expectation of genotoxicity. The absence of activity seen in the human lymphocyte micronucleus test clearly distinguishes the registered substance from other mineral fibres known to cause chromosome damage in mammalian cell assays in vitro. Hence it is concluded that no classification in respect of mutagenic activity is warranted.