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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Good quality, well documented study in accordance with relevant OECD, UKEMS and ICH guidelines.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UKEMS Guideline (Gatehouse et al 1990)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH-S2A (1995) and ICH-S2B (1997)
Deviations:
no
Principles of method if other than guideline:
Gatehouse DG, Wilcox P, Forster R, Rowland IR and Callander RD (1990) Bacterial mutation assays. In "Basic Mutagenicity Tests UKEMS Recommended Procedures". Report of the UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Part I Revised. Ed Kirkland DJ. Cambridge University Press, pp. 13-61
ICH-S2A (1995) “Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals”
ICH-S2B (1997) “Standard Battery for Genotoxicity Tests for Pharmaceuticals”
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrachloroauric acid
EC Number:
240-948-4
EC Name:
Tetrachloroauric acid
Cas Number:
16903-35-8
Molecular formula:
AuCl4.H
IUPAC Name:
tetrachloroauric acid
Test material form:
other: Orange liquid
Details on test material:
- Name: Tetrachloroauric acid
- CAS number 16903-35-8
- Batch number AY0014
- Physical state: Orange liquid
- Receipt date: 27 April 2012
- Storage: At 15-30°C protected from light
- Purity: Gold 41.78% w/w and chloride 29.87% w/w
- Expiry date: 2000 days from manufacture date of 11 March 2012.

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1 (with and without S9): 1.563 (without S9 only), 3.125, 6.250, 12.5, 25, 50, 100, 500 (with S9 only) µg/plate
Experiment 2 (without S9): 1.40 (TA100 and TA1537 only), 2.33, 3.89, 6.48, 10.8, 18, 30, 50 (TA98, TA1535 and TA102 only) µg/plate
Experiment 3 (with S9): 7.78, 12.96, 21.6, 36, 60, 100, 500 µg/plate
The test article concentrations stated do not correct for metal content but are expressed as Tetrachloroauric acid solution (as supplied).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that tetrachloroauric acid was soluble in purified water at concentrations of at least 50 mg/mL.
Controls
Untreated negative controls:
yes
Remarks:
Concurrent vehicle
Negative solvent / vehicle controls:
yes
Remarks:
Purified water
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Remarks:
A concurrent hydrochloric acid control was also used to determine the effects of pH on toxicity
Details on test system and experimental conditions:
- Bacteria: Bacteria (TA98, TA1535 and TA1537 originally sourced from the UK NCTC, TA100 and TA102 originally sourced from Covance Laboratories Inc., USA) were cultured in nutrient broth (TA98 and TA100 broth containing ampicillin, TA102 broth containing ampicillin and tetracycline) for approximately 10 hours in a shaking anhydric incubator at 37±1°C. All treatments were completed within 6 hours of the end of the incubation period. The strain characteristics of the master plates and vials of frozen culture were checked.
- Metabolic activation system: Male Sprague Dawley rats induced with Aroclor 1254 were used to prepare the mammalian liver post-mitochondrial fraction (S9) (Molecular Toxicology Incorporated, USA). Batches were checked by the manufacturer and frozen aliquots were thawed just before use.
- Method: Triplicate (test and positive controls) or quantuplicate (vehicle and hydrochloric acid controls) plates were prepared using 2.5 mL molten agar at 46±1°C with sequential additions of 0.1 mL bacterial culture, 0.1 mL test article solution or control, 0.5 mL 10% S-9 mix or buffer solution followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C in the dark for 3 days. As the results of the first experiment were negative, treatments in the presence of S9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution (reduced to 0.05 mL for positive controls), bacteria and S9 mix, were mixed together and incubated for 20 minutes at 37±1°C, before the addition of 2.5 mL molten agar at 46±1°C and plating following the normal plate incorporation procedure.
Evaluation criteria:
- Evaluation: Following incubation, the plates were examined for evidence of toxicity to the background lawn and revertant colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments). Individual plate counts were recorded and the mean and standard deviation of the plate counts for each treatment were determined. For valid data, the test article was considered to be mutagenic if: when assessed using Dunnett's test (which compared the counts at each concentration with the control), an increase in revertant numbers gave a statistically significant response (p less than or equal to 0.01) which was concentration related; and the positive trend/effects described above were reproducible.
- Controls: Vehicle and positive control counts were compared with the accepted normal ranges for the laboratory and considered acceptable if they fell within the historical 99% confidence intervals.
Statistics:
The presence of a concentration response was checked by non-statistical analysis up to limiting levels and counts at each concentration were compared with the control using the Dunnett's test.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Mutagenicity: No statistically significant increases in revertant numbers were observed when the results were analysed at the 1 % level using Dunnett's test and therefore, tetrachloroauric acid showed no evidence of any mutagenic activity in this assay system.
- Toxicity: Evidence of toxicity ranged from a diminution of the background bacterial lawn or a reduction in revertant numbers, to a complete killing of the test bacteria. In experiment 1, toxicity was observed at 12.5 µg/plate and above in strain TA1537 without S9; 25 µg/plate and above in strains TA98, TA100 and TA1535 without S9; 50 µg/plate and above in strain TA102 with S9 and at 100 µg/plate and above in all strains with S9. In experiment 2, toxicity was observed at 18 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 without S9; 50 µg/plate in strain TA102 without S9; 60 µg/plate and above in strains TA1537 and TA102 with S9; and 100 and/or 500 µg/plate in strains TA98, TA100 and TA1535 with S9.
- Controls: The controls were accepted as valid as they demonstrated correct strain and assay functioning. No clear evidence of toxicity was observed following the treatment of the HCl controls, indicating that toxicity observed following Tetrachloroauric acid treatment was unlikely to be due to the low pH of the test article.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, Tetrachloroauric acid did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium at treatments up to toxic concentrations in the absence and in the presence of a rat liver metabolic activation system (S9).
Executive summary:

Tetrachloroauric acid was tested for its ability to induce reverse mutations in an Ames assay conducted according to OECD guideline 471 and employing five strains of the bacterium Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). There was no mutation induced when multiple concentrations of Tetrachloroauric acid were tested up to toxic concentrations in the presence and absence of a rat liver metabolic activation system (S9).