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Description of key information

An MTD study (Rhodes 2015) was conducted to determine the maximum tolerated dose over short repeat-dose periods. The study was performed using dose levels unadjusted for the density of the test material, the adjustment being applied to the dose levels after completion of the study. The follow-up main study (Rhodes 2015), according to OECD422 guidelines was also conducted with dose levels unadjusted for the compound density. Fromulation analysis of solutions prepared for the main OECD422 study gave results that were outside the acceptable range of variation of such analyses. Consequently, the main OECD422 study was repeated (LPT 2017) in which dose levels were prepared on a w/v basis thus taking into consideration the density of the test material in the production of the dose concentrations.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
03 September 2013 to 12 November 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline followed
Version / remarks:
Although not designed for specific guidelines the study protocol is expected to meet the requirements of the relevant agencies.
Principles of method if other than guideline:
The objective of the study was to determine the maximum tolerated dose (MTD) of the test article, tetrachloroauric acid, following daily oral (gavage) administration to the rat. The toxicity of repeated daily administration at this dose level for 15 days was then assessed using naïve animals.
GLP compliance:
no
Remarks:
Study conducted in accordance with local Standard Operating Procedures (SOPs).
Limit test:
no
Species:
rat
Strain:
other: HsdHan: WIST
Details on species / strain selection:
Rats were supplied by Harlan, Bicester, UK
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Bicester, UK
- Age at study initiation: 9 (fixed-dose study) to 12 (MTD study) weeks old
- Weight at study initiation: 169.6 - 243.6 to 312.5 g (males) and 142.5 - 174.9 g (females) at Day 1 of predosing phase
- Housing: groups of up to 2 or 5 housed in cages that conform to the 'Code of practice for the housing and care of animals used in scientific procedures' (Home Office, London, 1989).
Wooden aspen chew blocks used as bedding, changed on a weekly basis.
- Diet (e.g. ad libitum): SQC Rat and Mouse Maintenance Diet No 1 (expanded) ad libitum
- Water (e.g. ad libitum): ad libitum

DETAILS OF FOOD AND WATER QUALITY: Each batch of diet was analysed and the water was periodically analysed.
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally by gavage.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily during the Fixed Dose phase and once every three days during the MTD phase. The control article and vehicle for the test article was purified water. Formulations were prepared using a v/v approach hence no account was taken for the density of the solution in the determination of the mg/kg dose level.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
MTD study: three days (Group 1, MTD), seven days (Group 5, MTD)
Fixed-dose study: 15 days
Frequency of treatment:
once daily
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
MTD, Group 1. Adjusted for density to 315 mg/kg
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
MTD, Group 5. Adjusted for density to 210 mg/kg
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Fixed Dose Phase, control
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Fixed Dose Phase, low dose. Adjusted for density to 168 mg/kg/day
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Fixed Dose Phase, high dose. Adjusted for density to 210 mg/kg/day
No. of animals per sex per dose:
MTD phase: 2
Fixed Dose phase: 4
Details on study design:
- Dose selection rationale: The purpose of this study was to determine a suitable range of dose levels that could be used in a repeat dose OECD422 study and an in vivo micronucleus assay. The starting dose of 150 mg/kg/day (315 mg/kg/day adjusted) was chosen on the basis of an acute toxicity study in rats (OECD 401 study, performed in 1993).
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations:
MTD Phase: Day –7 (randomisation body weight check). Before dosing on the day of each incremental change in dose level during the MTD phase.
Fixed Dose Phase: Twice weekly during the fixed dose phase. Day of (prior to) necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption recorded in g; calculated as g/animal/day.

HAEMATOLOGY, CLINICAL CHEMISTRY: ).5 mL terminal blood samples were taken into EDTA tubes and stored deep frozen for possible future analysis but were not analysed in the first instance.

URINALYSIS: Urine samples were taken for possible future analysis but were not analysed in the first instance. Urine samples were collected overnight and stored deep frozen;




Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A full macroscopic examination was performed. Appropriate tissues were processed and examined.

HISTOPATHOLOGY: No
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the MTD phase, severe effects observed at two animals (one male and one female) receiving 150 mg/kg/day on Days 1 to 3 inclusive. In the fixed dose phase, only minor clinical signs of mouth rubbing, paddling and salivation observed at 80 and 100 mg/kg/day.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Effects on body weight gain were noted in male and female rats at 150 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food intake was observed in male and female rats at 150 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Increased kidney weights, small increases in liver weights, slight organ weight changes to mesenteric lymph nodes (males and females), and adrenals (females only).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Significant macroscopic pathology kidney changes observed in two animals at 150 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Dose descriptor:
other: Maximum-Tolerated-Dose (MTD)
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
Adjusted for density to 210 mg/kg/day
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
organ weights and organ / body weight ratios
Critical effects observed:
yes
Lowest effective dose / conc.:
150
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The Maximum-Tolerated-Dose (MTD) for short term repeated administration was considered to be 100 mg/kg/day (210 mg/kg/day adjusted for density).
Executive summary:

The objective of the study was to determine the maximum tolerated dose (MTD) of the test article, Tetrachloroauric Acid, following daily oral (gavage) administration to the rat. This study was conducted in order to determine a suitable range of dose levels that could be used in a repeat dose OECD422 study and an in vivo micronucleus assay.

The test item was dosed by oral route to male and female rats once daily for three days (Group 1, MTD), seven days (Group 5, MTD) or fifteen days (Fixed Dose). The dose level of 150 mg/kg/day (315 mg/kg/day adjusted) showed significant toxicity in male and female rats with the kidney identified as a target organ. Dose levels of 80 and 100 mg/kg/day (168 and 210 mg/kg/day adjusted) were generally well-tolerated with no pathological changes noted in taget organs. Therefore, the MTD was determined to be 100 mg/kg/day (210 mg/kg/day adjusted).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
In-life dates: 05 August 2014 to 29 September 2014
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: HsdHan:WIST
Details on species / strain selection:
Rat is a readily available rodent species acceptable to the regulatory authorities and recommended for reproduction studies because of its reproductive characteristics.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Bicester, UK
- Age at study initiation: 10 to 12 weeks old
- Weight at study initiation: 243.5 to 312.5 g (males) and 181.2 to 240.5 g (females) at the start of dosing
- Housing: The animals were housed in groups of up to four (pre-pairing and post-pairing), one female with one male (pairing) or individually (mated females) in cages that conform to the 'Code of practice for the housing and care of animals used in scientific procedures' (Home Office, London, 1989). Bedding was provided on a weekly basis to each cage by use of clean Aspen wood shavings.
- Diet (e.g. ad libitum): SDS VFR1 (P), (Special Diets Services Ltd, Witham, UK) diet, provided ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY: Each batch of diet was analysed for specific constituents and contaminants. The water is periodically analysed for specific contaminants. No contaminants were present in water at levels which might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 45 to 65%
- Air changes (per hr): minimum 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: 05 August 2014 To: 29 September 2014
Route of administration:
oral: gavage
Details on route of administration:
The test article was administered orally by gavage.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily. The test article was formulated in purified water. The acidity was neutralised using NaOH with the formulation pH being checked for each preparation prior to administration. The target range was pH 6.9±1. The formulations were stored at room temperature (15 to 25°C) in a sealed container, protected from the light.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification of doses was performed by anaysis of gold in all the dosed groups by the inductively coupled plasma mass spectrometry (ICP-MS).
The stability of the gold in test article formulation at 1, 3 and 10 mg/mL were determined for 2 days when stored at 15 to 25°C, protected from light.
Samples (1 x 5 mL aliquot (random) from test formulations prepared for use on the first and last day of dosing were taken for analysis of achieved concentration. The mean of the homogeneity results was taken as the achieved concentration where the sampling occasion coincided.
Concentrations of gold in all the dosed groups at both time points, exceeded the ± 15.0 % acceptance limit. It was confirmed that concentrations of gold in the control samples were below the lower limit of quantification (<100 μg/mL). The variation in the analysed concentrations of the dose formulations was the principal reason for the study to be disregarded and subsequently repeated. A potential contributing factor was the absence of density adjustment in the preparation of the dose solutions and the application of the density factor (2.1) post dosing.
Duration of treatment / exposure:
The animals were dosed once daily to the males for 2 weeks prior to pairing, during the pairing period and until the day before necropsy and daily to the females for 2 weeks prior to pairing, during the pairing period and until Day 4 post-partum, inclusive. The females were allowed to litter and rear their offspring to Day 4 post-partum. Dosing was deferred if the animal was in or near parturition. The males were treated for 8 weeks prior to necropsy.
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
low dose. Adjusted for density to 21 mg/kg/day
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
intermediate dose. Adjusted for density to 63 mg/kg/day
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
high dose. Adjusted for density to 210 mg/kg/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels used were based on the results from a Maximum Tolerated dose study in which small increases in liver and kidney weights were recorded in animals treated at 80 or 100 mg/kg/day and the MTD was defined as 100 mg/kg/day (or 210 mg/kg/day when corrected for density).
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: weekly, before removal from the cage

DETAILED CLINICAL OBSERVATIONS: Yes
-Time schedule:
- health monitoring: twice daily (at the beginning and the end of the working day) for signs of ill health or overt toxicity.
- clinical examinations: detailed physical examination at weekly intervals
- post dosing observations: immediately after dosing and approximately 0.5, 1, 2 and 4 hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
Males were weighed weekly from Day 1 of the Predose phase (Day -7) to the day of necropsy, prior to necropsy, Females were weighed weekly from Day 1 of the Predose phase (Day -7) until confirmation of mating. They were weighed on Days 0, 7, 14 and 20 of Gestation and Days 1 and 4 of Lactation (post-partum).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 7 (day of necropsy) (males), day 5 post-partum (females)
- Anaesthetic used for blood collection: yes, isoflurane anaesthesia
- Animals fasted: Yes
- How many animals: 5 animals/group
- Parameters examined: haemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration. reticulocyte count, red cell distribution width, haemoglobin distribution width, total and differential white cell count, platelet count, platelet crit, mean platelet volume, platelet distribution width.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 7 (day of necropsy) (males), day 5 post-partum (females)
- Animals fasted: Yes
- How many animals: 5 animals/group
- Parameters checked: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, creatinine, urea, glucose

URINALYSIS: Yes
- Time schedule for collection of urine: week 7 (day of necropsy), total urine collected overnight
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: volume, colour, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and at once weekly intervals thereafter. In the last week of the dosing period (Week 8, before dosing) for males and on Day 4 post-partum (after pups were sent to necropsy and before dosing) for females, an assessment was made of sensory reactivity to stimuli, grip strength and motor activity.
- Dose groups that were examined: all animals in all dose groups
- Battery of functions tested: sensory activity, grip strength, locomotor activity, other: open field observations


Sacrifice and pathology:
GROSS PATHOLOGY: Yes, a full macroscopic examination was performed and all lesions were recorded. The uterus of all females was examined for the presence of implantation sites.

HISTOPATHOLOGY: Yes, tissues from parental animals were embedded in paraffin, sectioned and stained and slides were prepared. They were examined microscopically. In the first instance tissues were examined from the first 5 animals/sex for Groups 1 and 4. Due to findings seen in the kidneys and stomach (females only) histological preparations and slide examination for Groups 2 and 3 were also performed.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Minor post-dosing observations such as mouth rubbing, paddling and/or salivation were seen in all treated groups, with the frequency of observation generally increasing with increasing dose. These observations are generally considered to be a result of taste aversion rather than systemic toxicity.
Mortality:
no mortality observed
Description (incidence):
There was a single unscheduled death. Female No. 128 at 30 mg/kg/day was killed in a moribund state on Day 24 of the gestation phase. The animal was found to be pale and with abnormal stomach contents that included foetal remains. No microscopic examination was performed and the cause of morbidity is unknown.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain in Group 3 and 4 males showed a slight, dose-dependent decrease throughout the study. Mean body weight in females showed no clear dose-related trend.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Treated males showed a slight dose-dependent reduction in food intake compared to controls over the first 4 days of dosing only. Females were unaffected.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
effects observed, non-treatment-related
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no clear treatment related differences in functional or locomotor data seen in either sex, at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significant differences in mean adrenal gland, kidney and liver weights in males at 100 mg/kg/day only, when expressed relative to the overall average group terminal body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related macroscopic change was found in the kidneys of a single male at 100 mg/kg/day. This finding was irregular surface and was correlated microscopically with tubular nephropathy (moderate degree).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic observations were limited to the highest dosage group of 100 mg/kg/day and to the kidneys in both sexes and the stomach in females. In the kidneys, tubular nephropathy was seen in 4/5 males (minimal to moderate degree) and 2/5 females (minimal degree) at 100 mg/kg/day. Erosion/ulcer (minimal to slight degree) was noted in the pyloric region of the stomach of 2/5 females at 100 mg/kg/day. In the absence of concurrent stress-related findings, this finding is thought to be a direct effect of the test article administration.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
21 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
Dose level of 10 mg/kg/day adjusted for density
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Conclusions:
The NOAEL was considered to be 21.0 mg/kg/day.
Executive summary:

The repeat dose toxicity study with tetrachloroauric acid (TCA) was conducted according to OECD guideline 422.

The test item was administered daily by oral route (gavage) to rats at dose levels of 10, 30 and 100 mg test item/kg b.w./day alongside a control group. However, due to an error in calculation the density of the dose solution was not taken into consideration at the preparation of dose formulations stage. Therefore, dose levels following the correct were nominally 21.0, 63.0 and 210.0 mg/kg/day. Subsequent analysis of the formulations at designated time points indicated unacceptable variation in the gold content.

Daily administration of tetrachloroauric acid at a dose level of 100 mg/kg/day (210 mg/kg/day adjusted) elicited minor post-dosing observations of mouth rubbing, paddling and salivation, as well as slightly reduced food intake and body weight gain in males, increased incidence of small/missing/cannibalised/dead pups, organ weight changes in males to adrenal gland kidney and liver, changes to kidney pathology, and changes to stomach pathology in females. A dose level of 30 mg/kg/day (63 mg/kg/day adjusted) was associated with minor post-dosing observations of mouth rubbing, paddling and salivation, as well as slightly reduced body weight gain in males, and increased incidence of small/missing/cannibalised/dead pups. A dose level of 10 mg/kg/day (21.0 mg/kg/day adjusted) was associated with occasional minor post-dosing observations of mouth rubbing, paddling and salivation only.

The NOAEL was considered to be 10 mg/kg/day (21.0 mg/kg/day adjusted).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2016 to 26 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: CD / Crl:CD(SD)
Details on species / strain selection:
CD rats bred by Charles River Laboratories Germany GmbH were used for the test.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 92 days
- Weight at study initiation:
Males: 398.4 g - 497.5 g
Females: 240.0 g - 273.7 g
- Fasting period before study: no
- Housing: With the exception of the mating period, the males and females (F0-Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood was used for animal bedding.
- Diet (e.g. ad libitum): A certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH) offered ad libitum. Food residue was removed and weighed.
- Water (e.g. ad libitum): Tap water was offered ad libitum.
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: No contaminants above the limitations were noted in the drinking water or food. Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL. Additionally, water samples are taken once a year for bacteriological analyses. Samples of the food are analysed for contaminants based on EPA/USA4 by LUFAITL5 at least twice a year.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle (about 150 lux at approximately 1.5 m room height)

IN-LIFE DATES: From: 16.11.2016 To: 26.01.2017
Route of administration:
oral: gavage
Details on route of administration:
The test item formulations were administered orally at a constant dose volume of 10 mL/kg b.w./day per animal.
The dose of the test item was adjusted to the animal's actual body weight daily.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was supplied as liquid and suspended in the vehicle (purified water) to the appropriate concentrations for administration.The pH of the test item is approximately 1.5, therefore the test item was neutralised before dosing. As the density of the test item solution is high, the application formulation was prepared in a w/v procedure. The test item formulations were freshly prepared every day and the amount of the test item was adjusted to the animal's current body weight. The test item was diluted with purified water to the appropriate concentrations. The pH was adjusted to 6.9 with 0.1 M or 1 M sodium hydroxide (NaOH) solution.


VEHICLE: purified water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of gold in the test item, in control samples (aqua ad injectabilia), in samples of the application mixtures in course of the toxicological study and in carrier control samples (control group) was determined by ICP-OES.
For the analysis of the test item-vehicle formulations, samples of approximately 10 mL were taken at the following times and stored at ≤-20°C until shipment for analysis:
- At start of the dosing period and of dose change (300 mg/kg) - Analysis of stability and concentration: Immediately after preparation of the test item vehicle formulations as well as after 8 and 24 hours storage at room temperature (3 samples/group).
Towards the end of the treatment period (when the majority of animals are dosed) - Analysis of concentration: During treatment always before administration to the last animal/ group (1 sample/ group).

Sample preparation:
The frozen samples were defrosted at room temperature, transferred into a volumetric flasks and diluted with hydrochloric acid. The samples were subsequently analysed by ICP-OES.

Evaluation
The method used for this analysis measures the intensity of the spectral line at 267.594 nm. The results were obtained by comparison of the detected signal intensity of the samples with a linear calibration function generated with the external reference standard “ICP Multielement Standard 8”.
Duration of treatment / exposure:
- Adaptation: 13 days
- Pre-treatment period: 14 test days (TD 1 to TD 14)

- Start of treatment on test day 15
- Males: - 33 treatment days (sacrifice on TD 48)
- Females with litter: - 50 to 57 treatment days (sacrifice between TD 65 and 72)

Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
low dose
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
intermediate dose
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
high dose; dose was reduced to 375 mg/kg bw/day on the 2nd day of dosing and then to 300 mg/kg bw/day on the 3rd day of dosing
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on available toxicological data and a 14-day dose range finding study conducted with dose levels of 30, 100 or 300 mg/kg b.w./day
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes, behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity and mortalities were recorded.
- Time schedule:
Clinical observations: at least daily throughout the test period,
Mortality: twice daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum) and on day 4 and 13 postpartum. Body weights were recorded individually for each adult animal.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg b.w./day) was determined. Food residue or (total food left) was weighed and recorded

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION : Yes
- Time schedule for examinations: Drinking water consumption was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the premating period on
test day 29
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, animals were fasted overnight
- How many animals: 5 male and 5 female F0 animals randomly selected from each group
- Parameters examined: Haemoglobin content, Erythrocytes (RBC), Leucocytes (WBC), Differential blood count (relative and absolute), Reticulocytes, Platelets (PCT or PLT), Haematocrit value (PCV or HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the premating period on
test day 29
- Animals fasted: Yes, overnight
- How many animals: 5 male and 5 female F0 animals randomly selected
- Parameters examined: albumin, globulin, bile acids, bilirubin, chloesterol, creatinine, glucose, protein, blood urea, calcium, chloride, potasium, sodium, lactase dehydrogenase, Alanine aminotransferase (ALAT), alkaline phosphatase (aP), Aspartate aminotransferase (ASAT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at least 2 hours after dosing on test day 45 for the male rats and between lactation days 13 and 15 for the female rats.
- Dose groups that were examined: five males and five females randomly selected from each study group
- Battery of functions tested: sensory activity, grip strength, motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis.
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)
The respective calculations for the FISHER and Chi2 test in the histopathology report were performed using Provantis.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The high dose level of 450 mg/kg b.w. /day had to be stepwise reduced to 350 and 300 mg/kg b.w. /day after the 1st and 2nd administration, repectively, due to severe signs of clinical toxicity in the form of prone position and / or a reduced motility.

Salivation and piloerection were observed at dose level of 300 mg test item/kg b.w./day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No premature death was noted in the control group and no test item-related premature death was noted in the treatment groups (50, 150 or 450/375/300 mg test item/kg b.w./day).
One male animal of the intermediate dose group died prematurely by misgavage (found dead in the morning of test day 44).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight was noted for the female animals of the high dose group on test day 22, at theend of the gestation and lactation periods. No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related changes or changes that were of toxicological relevance were noted in food consumption for the rats of the low, the intermediate and the high dose group (50, 150 or 450/375/300 mg test item/kg b.w./day).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in the consumption of drinking water was noted for the male and female rats treated with 50, 150 or 450/375/300 mg test item/kg b.w./day by visual appraisal.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 450/375/300 mg test item/kg b.w./day a statistically significant decrease (p ≤ 0.05/0.01) was noted for the haemoglobin concentration, the number of red blood cells and the haematocrit value.
A statistically significant increase (p ≤ 0.01) was noted for the number of reticulocytes and the number of neutrophilic granulocytes.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slight but statistically significant changes were noted at the high dose level for the albumin and globulin concentrations and their related parameter (albumin/globulin ratio, protein (total) concentration). A statistically significant decrease was noted for the activity of the alkaline phosphatase of the male and female animals of the high dose group. Statistically significant changes were further noted for the calcium concentration,
the glucose concentration and the sodium/potassium ratio for the male or female animals at the intermediate or the high dose level. These changes were considered to be not test item-related,
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test item-related influence on the fore- and hindlimb grip strength or on the spontaneous motility was noted for the male and female animals in any of the treatment groups (50, 150 or 450/375/300 mg test item/kg b.w./day).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males
Increased absolute and relative kidney weights (left and right) were noted for the male animals of the high dose group (450/375/300 mg test item/kg b.w./day) (between 15.9 % and 25.7% above the values of the control group; p ≤ 0.01).
Females
At 150 mg test item/kg b.w./day slightly increased absolute and relative kidney weights were noted for the female animals (between 5.5 % and 11.1 % above the values of the control group; statistically not significant or significant at p ≤ 0.01). At 450/375/300 mg test item/kg b.w./day an increase was noted in the absolute and relative organ weights of the left and right kidneys of the female animals (between 15.9 % and 25.7% above the values of the control group; p ≤ 0.01).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females
No obervations were noted that were of toxicological relevance in any of the test item treated groups.
However, discolourations or coatings of the stomach mucosa were noted in 2 of 10 male and in 2 of 10 female animals of the high dose group (450/375/300 mg test item/kg b.w./day), as a result of test item depositions. As no correlating microscopic findings were noted, these observations were not considered as adverse or of toxicological relevance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males and females
At 450/375/300 mg test item/kg b.w./day, kidney lesions were noted for all examined male and female animals in the form of increased basophilic tubules in the renal cortex and a degeneration of proximal and distal tubules (5 of 5 animals).
Furthermore, one kidney with a focal necrosis (moderate) of the tubules in the cortex was noted for 1 of 5 male animals and an increased mineralization in the renal cortex of both kidneys was noted for 3 of 5 female animals of the high dose group.
Degenerated proximal and distal tubules were noted for the kidneys of all examined male and female animals (5 of each sex) of the intermediate dose group (150 mg test item/kg b.w./day). Additionally, 2 of 5 female animals of the intermediate dose group revealed increased basophilic tubules.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
water consumption and compound intake
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The NOAEL for systemic toxicity was detemined to be 50 mg test item/kg b.w./day, p.o.
Executive summary:

The repeat dose toxicity study with Tetrachloroauric acid (TCA) was conducted according to OECD guideline 422.

The test item was administered orally by gavage to rats at dose levels of 50, 150 or 450/375/300 mg test item/kg b.w./day. The high dose level had to be stepwise reduced after the 1st and 2nd administration, due to severe signs of clinical toxicity in form of prone position and / or a reduced motility.

Changes in the behaviour or the external appearance that were referred to the dose level of 300 mg test item/kg b.w./day were salivation and piloerection. Further observations at 300 mg test item/kg b.w./day were in the form of a reduced body weight that was noted for the female animals together with changes in several haematological parameters. The examinations at termination revealed increased absolute and relative kidney weights for the female animals at 150 mg test item/kg b.w./day and for the male and female animals at 300 mg test item/kg b.w./day. The increased kidney weights could be correlated with histopathological kidney changes (e.g. increased basophilic tubules, degenerated proximal and distal tubules) that were noted at 150 and 300 mg test item/kg b.w./day.

Based on the results, the NOAEL for systemic toxicity was established to be 50 mg TCA/kg b.w. /day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The Key study is a Klimisch 1 (reliable without restrictions), guideline study performed in accordance with GLP conditions.
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The oral repeated-dose toxicity NOAEL was determined to be 50 mg/kg/day (over 33-days in male rats and up to 57 days in females with a litter) in the key repeat-dose toxicity study, and the lowest determined effect level was 150 mg/kg/day based on histopathological changes seen in the kidneys of the majority of animals at this dose level. The guidance dose ranges for Specific Target Organ Toxicity by repeat oral exposure (STOT-R) would be Category 1, <10-30 mg/kg/day, or Category 2, 10/30 – 100/300 mg/kg/day, depending on the duration of exposure (90-28 days respectively). Considering the lowest effect level of 150 mg/kg/day in the source study was associated with a slight degenerative effect on the rat kidney after 33-57 days of exposure, it is clear that this dose level falls outside the Category 1 criteria (i.e. the effective dose level exceeds the highest threshold >30 mg/kg/day). However, for Category 2 classification, the minimal effect level of 150 mg/kg/day exceeds the 90-day threshold of 100 mg/kg/day but is within the 28-day range of 30-300 mg/kg/day. Since the renal toxicity observed at the dose level of 150 mg/kg/day falls within the guideline criteria considered indicative of relevant toxic effects, Category 2 classification for STOT-RE classification is considered to be applicable.