Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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

Test material

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:
liquid

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

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

Applicant's summary and conclusion

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.