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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus 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:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Physical state: Orange liquid
- Expiration date of the lot/batch: 02 September 2018
- Storage condition of test material: Stored at 15-25°C and protected from light in a tightly sealed container.

Test animals

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

Administration / exposure

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).
Doses / concentrations
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.

Examinations

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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.

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