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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A reverse mutation test was conducted in five histidine requiring strains of Salmonella typhimurium (Ames Test; OECD471)) under Annex VII of REACH regulations. As a consequence of a negative result in the Ames test, the Annex VIII in vitro tests were undertaken. To examine the potential to cause chromosome damage, a study of the induction of micronuclei in cultured human peripheral lymphocytes (OECD487) was performed. A positive result was obtained in this study.

Link to relevant study records

Referenceopen allclose all

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.
Reason / purpose for cross-reference:
reference to same study
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
Target gene:
Histidine operon
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.
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.
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'.
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).

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: chromosome damage – micronuclei
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
other: OECD 487 (In vitro mammalian cell micronucleus test)
Deviations:
no
Principles of method if other than guideline:
OECD (2010) Genetic toxicology: OECD guideline for the testing of chemicals. Guideline 487: In vitro mammalian cell micronucleus test
The test methodology is also based on accepted scientific/regulatory principles described in current guidelines for clastogenicity test in vitro.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Chromosome damage by measuring micronuclei
Species / strain / cell type:
primary culture, other: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: Blood from two healthy female volunteers from a panel of donors at the testing laboratory was used. No volunteer was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All volunteers are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication (contraceptive pill excluded). The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 2 hours.
Metabolic activation:
with and without
Metabolic activation system:
Liver post-mitochondrial fraction (S9) obtained from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
ANALYSED
- Experiment 1 (3+21 without S9): 0 (purified water), 40.00, 80.00, 140.0 µg/mL (plus positive control MMC 0.80 µg/mL)
- Experiment 1 (3+21 with S9): 0 (purified water), 40.00, 70.00, 95.00, 125.0 µg/mL (plus positive control CPA 12.50 µg/mL)
- Experiment 1 (24+24 without S9): 0 (purified water), 30.00, 45.00, 60.00 µg/mL (plus positive control VIN 0.04 µg/mL)
- Experiment 2 (3+21 without S9): 0 (purified water), 30.00, 40.00, 60.00, 120.0 µg/mL (plus positive control MMC 0.80 µg/mL)
- Experiment 3 (3+21 without S9): 0 (purified water), 40.00, 80.00, 140.0, 200.0 µg/mL (plus positive control MMC 0.80 µg/mL)
- Experiment 3 (24+24 without S9): 0 (purified water), 30.00, 40.00, 45.00, 60.00 µg/mL (plus positive control VIN 0.10 µg/mL)

TESTED
- Range finder (3+21 and 24+24, with and without S9): 0, 12.33, 20.55, 34.24, 47.07, 95.12, 458.5, 264.2, 440.4, 734.0, 1223, 2039, 3398 µg/mL
- Experiment 1 (3+21 without S9): 0, 20.00, 40.00, 60.00, 80.00, 100.0, 120.0, 140.0, 160.0, 180.0, 200.0, 250.0, 300.0, 400.0 µg/mL
- Experiment 1 (3+21 with S9): 0, 20.00, 40.00, 60.00, 70.00, 80.00, 90.00, 95.00, 100.0, 110.0, 125.0, 150.0, 200.0 µg/mL
- Experiments 1 and 3 (24+24 without S9): 0, 5.000, 10.00, 20.00, 25.00, 30.00, 35.00, 40.00, 45.00, 50.00, 60.00, 80.00, 100.0 µg/mL
- Experiments 2 and 3 (3+21 without S9): 0, 10.00, 20.00, 30.00, 40.00, 50.00, 60.00, 80.00, 100.0, 120.0, 140.0, 160.0, 200.0 µg/mL
- Positive controls: Mitomycin C: 0.60, 0.80 µg/mL, Cyclophosphamide: 6.25, 12.50 µg/mL, Vinblastine: 0.04, 0.06, 0.08 (0.10, 0.12 experiment 3 only) µg/mL

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 up to at least 50.00 mg/mL.
- Preparation: Stock solutions were prepared by vortex mixing under subdued lighting before being membrane filter-sterilised (0.2 µm pore size) and subsequently diluted with purified water. The test article solutions were protected from light and used within approximately 4 hours of initial formulation.
Untreated negative controls:
yes
Remarks:
(concurrent vehicle)
Negative solvent / vehicle controls:
yes
Remarks:
Purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Vinblastine
Details on test system and experimental conditions:
MICRONUCLEUS ASSAY
- 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. The rat liver was mixed with 180 mg/mL glucose-6-phosphate, 25 mg/mL β-Nicotinamide adenine dinucleotide phosphate, 150 mM potassium chloride in the ratio 2:1:1:1 and added to each cell culture to acheive the final test article concentration in 10 mL so that the final liver homogenate concentration was 2%. Cultures without S9 received an equivalent volume of 150 mM KCl.
- Blood cultures: 0.4 mL (range finding and experiment 1) or 0.8 mL (experiments 2 and 3) of pooled heparinised blood into 8.1 mL (range finding and experiment 1) or 7.7 mL (experiments 2 and 3) HEPES-buffered RPMI medium (pre-warmed to 37°C) containing 10 % (v/v) heat inactivated foetal calf serum and 0.5 2% penicillin/streptomycin along with approximately 2 % phytohaemagglutinin. Cultures were incubated with continuous rocking for 48 hours at 37°C.

FLUORESCENCE IN SITU HYBRIDISATION (FISH)
- Method: Slides were freshly prepared from stored fixed cell pellets from two (24+24 hour) or three (3+21 hour) test article concentrations which had clearly demonstrated statistically significant increases in the frequency of MNBN cells. Slides were aged at room temperature overnight prior to hybridisation with pan centromeric human DNA probes. Slides were pre-treated in saline sodium citrate (SSC)/0.5% igepal (pH 7.0) at 37°C for 15 minutes before dehydration by a series of 1 minute ethanol washes (70%, 85% and 100% ethanol) prior to drying at room temperature. The sample and FISH probe were denatured at 75°C for 5 minutes using the StatSpin® ThermoBriteTM then hybridised overnight in a humidified chamber at 37°C. Samples were washed with and without coverslips then dehydrated using ethanol washes. Slides were allowed to air dry, mouted in DAPI antifade and stored at 2-8°C until analysis.
Evaluation criteria:
MICRONUCLEUS ASSAY
- Slide analysis: Binucleate cells were only included in analysis if the cytoplasm remained essentially intact and the daughter nuclei were of approximately equal size. Micronuclei were recorded if the micronucleus had the same staining characteristics and a similar morphology to the main nuclei, any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.
- Acceptance criteria: The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures, the frequency of MNBN cells in vehicle controls fell within the current historical vehicle control (normal) ranges, the positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei and a minimum of 50% of cells in vehicle control cultures had gone through at least one cell division at the time of harvest.
- Evaluation criteria: For valid data, the test article was considered clastogenic and/or aneugenic if a statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed, an incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed and a concentration-related increase in the proportion of MNBN cells was observed.

FLUORESCENCE IN SITU HYBRIDISATION (FISH)
- Slide analysis: Fluorescence microscopy using appropriate filters. The mechanism of action was determined from the proportions of centromere positive (one or more fluorescent signals) and centromere negative (no fluorescent signals) micronuclei (per concentration).
- Acceptance criteria: Micronuclei in the cultures treated with the reference clastogen (MMC) were predominantly centromere-negative and the micronuclei in cultures treated with the reference aneugen (VIN) were predominantly centromere-positive.
Statistics:
The number of micronuclei per binucleate cell were obtained and recorded. A binomial dispersion test was used to establish acceptable heterogeneity between replicates using the proportions of MNBN (binucleate cells with micronuclei) cells. The proportion of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test with a significance probability of p ≤ 0.05.
Species / strain:
primary culture, other: human peripheral blood lymphocytes
Metabolic activation:
with
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
primary culture, other: human peripheral blood lymphocytes
Metabolic activation:
without
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Results: Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the current historical vehicle control ranges.
- pH: No marked changes in pH were observed as compared to the concurrent vehicle controls for experiments 1, 2 and 3.
MICRONUCLEUS ASSAY
- Validity: The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures in Experiments 1-3 with the exception of the 24+24 hour treatments without S9 in Experiment 3 (p ≤ 0.01), though positive data was expected in this test.
- Controls: The vehicle controls fell within the normal range, with the exception of marginal sporadic increases in the 3+21 hour treatments without S9 in Experiments 1 and 3 (1.1% compared to the 0-1% normal range). Two further vehicle replicate cultures in both experiments confirmed these to be isolated increases which did not affect the validity of the data. The positive control chemicals induced statistically significant MNBN cell frequencies that exceeded the current historical vehicle control ranges. A minimum of 50% of cells had gone through at least one cell division in vehicle control cultures at the time of harvest with the exception of one replicate in the 24+24 hour treatment without S-9 in Experiment 3, though all four cultures were analysed as the other three replicates exceeded 50%.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Micronucleus Experiments – Results summary

Treatment

Concentration (mg/mL)

Cytotoxicity (%)A

Mean MNBN cell frequency (%)

Statistical significance

3+21 hour -S-9 (Experiment 1)

VehicleB

-

0.8G

-

40

6

1.25

p0.05

80

34

2.2

p ≤ 0.001

140

49

1.75

p ≤ 0.001

Positive controlC

ND

13.7

p0.001

3+21 hour +S-9 (Experiment 1)

VehicleB

-

0.6G

-

40

5

0.85

NS

70

17

0.85

NS

95

37

1.2

p0.05

125

54

1.1

p0.05

Positive controlD

ND

2.85

p0.001

24+24 hour -S-9 (Experiment 1)

VehicleB

-

0.3H

-

30

0

0.9

p0.01

45

14

1.85

p0.001

60

36

2.45

p0.001

Positive controlE

ND

3.25

p0.001

3+21 hour -S-9 (Experiment 2)

VehicleB

-

0.5G

-

30

0

0.85

NS

40

8

0.55

NS

60

26

0.65

NS

120

42

0.4

NS

Positive controlC

ND

7.95

p0.001

3+21 hour -S-9 (Experiment 3)

VehicleB

-

0.53G

-

40

4

0.8

NS

80

18

1.85

p0.001

140

24

1.45

p0.001

200

63

2.4

p0.001

Positive controlC

ND

18.3

p0.001

24+24 hour -S-9 (Experiment 3)

VehicleB

-

0.43H

-

30

0

1.4

p0.001

40

0

1.8

p0.001

45

0

0.9

p0.05

60

0

1.65

p0.001

Positive controlF

ND

18.2

p0.001

 

A - Based on respiration index

B - Vehicle control was purified water

C - Positive control: Mitomycin C 0.80 µg/mL

D - Positive control: Cyclophosphamide 12.50 µg/mL

E - Positive control: Vinblastine 0.04 µg/mL

F - Positive control: Vinblastine 0.10 µg/mL

G - Historical control range (95th percentile calculated range): 0.10 - 1.00 %

H - Historical control range (95th percentile calculated range): 0.10 - 1.40 % (historical control range was calculated on 24 +0 hour without S9 treatments. Range included as a guide only)

NS - Not signficant

ND - Not determined

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

Tetrachloroauric acid induced micronuclei in cultured human peripheral blood lymphocytes following 3+21 hour and 24+24 hour treatment in the absence of a rat liver metabolic activation system (S9), when tested up to the limit of solubility in the test system. Subsequent mechanistic analysis demonstrated that micronuclei were generated via a predominantly clastogenic mechanism. Tetrachloroauric acid did not induce biologically relevant increases in the frequency of micronuclei following 3+21 hour treatment in the presence of S9, when tested up to cytotoxic concentrations.
Executive summary:

Tetrachloroauric acid was tested for its ability to induce micronuclei in cultured human peripheral blood lymphocytes according to OECD guideline 487 in the presence and absence of rat liver metabolic activation systems (S9). There was no mutation induced when multiple concentrations of Tetrachloroauric acid were tested up to toxic concentrations in the presence of a rat liver metabolic activation system (S9). Clastogenic mutation was induced when multiple concentrations of Tetrachloric acid were tested up to the limit of solubility of the test system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Due to the positive result in the in vitro micronucleus assay (OECD487), under the REACH regulations at the time, an in vivo test was required to be carried out and this was done under the wording of Annex VIII. Consequently, a rat bone marrow micronucleus assay (OECD474) was conducted to examine the potential to induce micronuclei in the polychromatic erythrocytes of the bone marrow of treated rats.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restrictions. GLP-compliant study conducted according to guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Han Wistar Crl:WI (Han) rats
- Source: Charles River (UK) Ltd., Margate, UK
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: 236 to 275 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Wire topped, solid bottomed cages with clean suitable wood bedding (Aspen).
- Diet (e.g. ad libitum): SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham), ad libitum.
- Water (e.g. ad libitum): Mains water, ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 23 °C
- Humidity (%): 45 to 52 %
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 03 August 2014 To: 06 August 2014
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Purified water
- Amount of vehicle (if gavage or dermal): 10 mL/kg bodyweight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test article was weighed and the appropriate volume of vehicle was added. The formulation was magnetically stirred and pH monitored until a stable reading was obtained. Sodium hydroxide solution was gradually added to adjust the pH to approximately 6.9±1. Formulations were freshly prepared prior to each dosing occasion by formulating Tetrachloroauric acid in purified water to give the test concentrations.
Duration of treatment / exposure:
Two consecutive days.
Frequency of treatment:
Two administrations at 0 hours (Day 1) and 24 hours (Day 2).
Post exposure period:
Animals observed until 48 hours after first exposure (Day 3).
Remarks:
Doses / Concentrations:
25, 50 and 100 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
6 male rats per treatment replicate and 3 male rats per control replicate.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: Oral gavage, 10 mL/kg dose volume.
- Doses / concentrations: Single administration of 20 mg/kg cyclophosphamide in 0.9 % saline at 24 hours.
Tissues and cell types examined:
Bone marrow sampled 24 hours after the last dose administration (Day 3, equivalent to 48 hours).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Animals at all treatment concentrations were analysed.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): One femur was removed and bone marrow isolated from all animals at necropsy.

DETAILS OF SLIDE PREPARATION: Bone marrow was flushed from the marrow cavity with foetal bovine serum into centrifuge tubes. The samples were filtered through cellulose columns and additional serum was added to the sample tubes and loaded onto the columns. Once filtered, the bone marrow cells were pelleted by centrifugation and the supernatant aspirated and discarded. Foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube, and from each tube one drop of suspension was placed on the end of each of two uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were air-dried, then fixed in absolute methanol and rinsed several times in distilled water. One slide per animal was immediately stained in acridine orange made up in 0.1 M phosphate buffer. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis.

METHOD OF ANALYSIS: Scoring was carried out using fluorescence microscopy at an appropriate magnification.

OTHER:
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. At points that were significant the group mean MN PCE value was outside the 95% historical vehicle control range
3. At the same dose levels the distribution of MN PCE in the majority of animals exceeded the laboratory’s historical vehicle control data
4. A dose-response trend in the proportion of MN PCE was observed.
Statistics:
For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation. The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p≤0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
Sex:
male
Genotoxicity:
negative
Remarks:
Tetrachloroauric acid did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to100 mg/kg/day under the experimental conditions employed.
Toxicity:
no effects
Remarks:
No clinical signs of toxicity were observed in any animal following treatments with vehicle, Tetrachloroauric acid (at 25, 50 or 100 mg/kg/day) or the positive control (CPA).
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Male and female rats were dosed daily for three days with 150 mg/kg/day or 7 days at 100 mg/kg/day Tetrachloroauric acid.
- Clinical signs of toxicity in test animals: At the dose level of 150 mg/kg/day toxicity in the form of severe clinical signs (lethargy, reduced activity, mouth rubbing and raised fur were seen in most animals), reduced food intake and body weight gain was observed. Significant macroscopic pathology kidney changes and increased kidney weights were also noted at necropsy and 2/4 animals had to be killed on humane grounds. A dose level of 100 mg/kg/day was generally well-tolerated with minor clinical signs of mouth rubbing, paddling and salivation observed.
- The dose levels quoted in this study when adjusted for density are equivalent to 315 and 210 mg/kg/day.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): For dose groups of 25, 50 and 100 mg/kg/day the %MN PCE values were 0.21%, 0.13% and 0.14% respectively compared to the concurrent vehicle control value of 0.17% MN PCE. These data indicate no evidence of a test article related effect on MN induction.
- The dose levels quoted in this study were not adjusted for density and are therefore a factor of 2.1 lower than the dose levels used in the preliminary MTD study.
Conclusions:
Interpretation of results (migrated information): negative
Tetrachloroauric acid did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 100 mg/kg/day (considered a suitable estimate of the maximum tolerated dose (MTD)) under the experimental conditions employed. However, this dose level is much lower than the estimated MTD (210 mg/kg/day adjusted for density) from the preliminary study.
Executive summary:

The effects of tetrachloroauric acid on chromosome aberration was examined in a bone marrow micronucleus test with Han Wistar rats. Male rats were dosed with 10 mL/kg bodyweight of tetrachloroauric acid in purified water by oral gavage at nominal concentrations of 25, 50 and 100 mg/kg/day. Two applications were made on consecutive days (Days 1 and 2) and rats were necropsied on Day 3. Observations of clinical toxicity and body weights were made throughout the test and bone marrow were analysed upon test completion. Tetrachloroauric acid did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 100 mg/kg/day (considered a suitable estimate of the maximum tolerated dose (MTD)) under the experimental conditions employed. No clinical signs of toxicity or notable effects on treatment on individual bodyweights were observed. This study is reliable without restriction as it was performed according to guideline and GLP. However, the high dose level of 100 mg/kg/day is much lower than the estimated MTD (210 mg/kg/day adjusted for density) from the preliminary study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Tetrachloroauric acid was tested for its ability to induce reverse mutations in a screening and definitive Ames assay (McGarry 2012a, McGarry 2012b) in five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). Three experiments were carried out with 7 to 8 test concentrations. There was no mutation induced at concentrations up to toxic concentrations in the presence and absence of rat liver S9 metabolic activation. Cytotoxicity was observed in various strains at various concentrations with and without metabolic activation. The definitive test was a GLP, guideline study following OECD 471 and is suitable for use as a key study.

In an in vitro experiment for chromosomal damage, cultured human peripheral blood lymphocytes were exposed to 12 to 13 concentrations of tetrachloroauric acid with and without activation for 24+24 and 3+21 hours. Tetrachloroauric acid was found to induce micronuclei in the absence of rat liver S9 metabolic activation when tested up to the limit of solubility (50.00 mg/mL), however a negative result was observed in the presence of metabolic activation when tested up to cytotoxic concentrations (Watters 2013). This was a GLP, guideline study following OECD 487 and is suitable for use as a key study.

In an in vivo study on chromosome aberration, male Han Wistar rats were orally gavaged with nominal concentrations of 25, 50 and 100 mg/kg/day tetrachloroauric acid on two consecutive days (Whitwell 2015). No evidence of micronuclei induction was observed in the polychromatic erythrocytes of the bone marrow at doses up to 100 mg/kg/day. No clinical signs of toxicity or notable effects on bodyweight were observed. This was a GLP, guideline study following OECD 474 and is suitable for use as a key study. The high dose level of 100 mg/kg/day was subsequently found to be below the likely MTD which was more likely to be in the range 210 -300 mg/kg/day. However, analysis of plasma and urine from rats on the MTD study for gold content demonstrated significant exposure but not dose dependent at nominal dose levels of 80 or 100 mg/kg/day (density adjusted levels 168 and 210 mg/kg/day). Therefore it was concluded that this in vivo chromosome aberration study was conducted at dose levels which provided significant exposure to the animals.

In a recombination assay with bacterium Bacillus subtilis strains H17 and M45, tetrachloroauric acid produced a negative result for evidence of DNA damage and repair at concentrations between 0.005M and 0.5M (Kanematsu et al. 1980). This study is used as supporting evidence as it was a non-GLP and non-guideline study with limitations in reporting, published in a peer-reviewed journal.

Overall, there is no consistent evidence of genotoxicity below the cytotoxic concentration for tetrachloroauric acid.


Justification for selection of genetic toxicity endpoint
By applying a weight-of-evidence approach, after consideration of the predominantly negative test results of highly reliable in-vitro and in-vivo genotoxicity assays, the overall conclusion is reached that no genotoxicity needs to be expected from exposure to tetrachloroauric acid.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In consideration of the predominantly negative test results in highly reliable genotoxicity assays, no genotoxicity needs to be expected from exposure to tetrachloroauric acid. In consequence, no classification is required.