Zirconium disilicide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Salmonella Mutagenicity Tests: II. Results From the Testing of 270 Chemicals

Author:

Mortelmans K. et al.

Year:

1986

Bibliographic source:

Environmental Mutagenesis Volume 8, Supplement 7, 1-119

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

see box "Principles of method if other than guideline"

Principles of method if other than guideline:

Study conducted similar to OECD 471. However, only four Salmonella typhimurium strains were used.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

25399-81-9

Molecular formula:

Cl2H12O7Zr

Specific details on test material used for the study:

- Name of the test material (as cited in study report): Zirconium oxychloride, 6H2O
- CAS number: 25399-81-9
- Purity: technical grade
- Source: Fluka Chemical Co.

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Cells were obtained from Dr. Bruce Ames, University of California, Berkeley, USA.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat and hamster metabolic activation systems

Test concentrations with justification for top dose:

Test concentrations: 10, 33, 100, 333, 1000, 3333, 6666 µg/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol (95%)

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without S9

Controls 2

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all four strains, with S9

Controls 3

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100, without S9

Controls 4

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

TA98, without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

EXPERIMENTAL PERFORMANCE
All chemicals were assayed for mutagenicity in the preincubation assay [Haworth et al., 1983]. To each of 13 x 100-mm test tubes maintained at 37 °C were added in the following order: 0.5 mL of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 mL of the overnight culture, and 0.05 mL of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37 °C for 20 min, at whichtime 2.5 mL (EGG) or 2.0 mL (CWR, SFU) of molten (45 °C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 mL of minimal glucose bottom agar [Vogel and Bonner, 1956] in 15 x 100-mm plastic petri dishes and Fisher Scientific plates. When the top agar had solidified, the
plates were inverted and incubated at 37 °C for 48 hr.

DURATION
- Preincubation period (Experiment II): 20 min at 37 °C
- Exposure duration: 48 h in the dark at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/dose level per experiment. All experiments were repeated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by appearance of his negative pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn.

Evaluation criteria:

The criteria used for data evaluation were the same as those described previously [Haworth et al., 1983] and are summarized as follows: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) nonmutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity. The initial determination of mutagenic, nonmutagenic, or equivocal was made by the testing laboratory; the final determination was made by the project officer (E.Z.). The chemicals were decoded by the chemical repository (Radian Corporation) only after the mutagenicity or nonmutagenicity of the chemicals had been determined.

Results and discussion

Test results

Key result

Species / strain:

other: TA1535, TA1537, TA100 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

For detailed results please refer to table 1 in box "Any other information on results incl. tables"

Remarks on result:

other: non-mutagenic

Any other information on results incl. tables

Table 1: Summary of Results
Concentration  µg/plate		TA100						Concentration  µg/plate		TA1535
		without S9		with 10% hamster S9		with 10% rat S9				without S9				with 10% hamster S9				with 10% rat S9
		Mean	SEM	Mean	SEM	Mean	SEM			Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
Negative Control		142	4.6	127	1	150	5.5	Negative Control		31	2.6	9	1.3	34	2.4	24	3.5	34	3.3	29	1.5
Positive Control		268	19.3	1572	41.5	742	48.2	Positive Control		344	6.9	175	23.2	505	12.5	326	15.2	223	19.9	114	6.1
10		-	-	-	-	-	-	10		28	5.2	-	-	36	2.3	-	-	45	5.8	-	-
33		-	-	-	-	-	-	33		29	3.5			36	3.2	-	-	39	2.2	-	-
100		125	8.2	158	9.5	136	4.5	100		30	2.4	22	3.0	41	3.5	33	2.3	42	6.6	30	1.0
333		108	10.9	169	10.5	126	7.1	333		30	1.0	29	6.4	38	6.1	37	3.1	35	3.7	33	3.6
1000		116	13.1	156	7.0	134	3.7	1000		30p	1.0	20	4.2	30p	0.3	35	4.9	38p	6.8	31	3.2
3333		149p	13.5	144p	14.0	77p	38.8	3333		-	-	26p	3.8	-	-	0p	0.0	-	-	29p	6.8
6666		48p	24.0	126p	5.2	35p	35.3	6666		-	-	0p	0.0	-	-	0p	0.0	-	-	0p	0.0

p= precipitation present in plates

Table 1 continued: Summary of Results
Concentration  µg/plate		TA1537						TA98
		without S9		with 10% hamster S9		with 10% rat S9		without S9		with 10% hamster S9		with 10% rat S9
		Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
Negative Control		7	2.9	7	0.7	12	3.2	25	0.0	32	0.9	30	3.5
Positive Control		109	8.7	536	34.9	197	7.6	793	25.9	1385	100.5	556	36.2
10		-	-	-	-	-	-	-	-	-	-	-	-
33		-	-	-	-	-	-	-	-	-	-	-	-
100		8	0.9	13	4.2	7	1.2	21	4.3	34	2.7	30	0.6
333		8	1.9	8	1.9	9	2.6	28	1.5	34	2.6	32	4.2
1000		8	1.8	12	2.0	9	2.2	21	4.6	32	5.8	27	4.1
3333		9p	1.8	7p	2.5	13p	0.9	19p	1.5	30p	3.0	28p	2.2
6666		0p	0.0	0p	0.0	0p	0.0	0p	0.0	0p	0.0	0p	0.0

p= precipitation present in plates

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item can be considered as non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria conducted similar to OECD test guideline 471, strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium were exposed to Zirconium oxychloride hexahydrate in 95% ethanol at concentrations of 10, 33, 100, 333, 1000, 3333 and 6666 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains. Therefore, Zirconium oxychloride hexahydrate is considered to be non-mutagenic in this Salmonella typhimurium reverse gene mutation assay.