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Genetic toxicity: in vitro

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
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%)
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Potassium chloride
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, without S9
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
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
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.