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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 September 2014 - 27 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant, guideline study available as an unpublished report. No restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Potassium dicyanoaurate
- Molecular formula: KAu(CN)2
- Physical state: White powder
- Storage condition of test material: Stored at 10 to 25 °C

Test animals

Species:
rat
Strain:
other: CD® Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 68 days
- Weight at study initiation: Males (MS): 337.1-369.4 g; Females (MS): 213.0-263.8 g; and Females (TX): 202.1-266.6 g. The body weight range did not exceed 20% of the mean weight for each sex at the time of selection.
- Fasting period before study: no
- Housing: MAKROLON cages type III plus (39 x 23 x 18 cm) with granulated textured wood for animal bedding
- Diet (e.g. ad libitum): Certified commercial diet (ssniff® R/Z V1324) provided ad libitum
- Water (e.g. ad libitum): Tap water provided ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was supplied as powder and suspended in tap water to the appropriate concentrations for administration. Test item formulations were prepared each day.

VEHICLE
- Vehicle: Tap water
- Amount of vehicle (if gavage): 5 mL/kg b.w/day
- Frequency of administration: once daily
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle mixtures, samples of approx. 10 mL were taken at the following times and stored at ≤ -20°C until shipment to Allessa GmbH, Germany.
- At study initiation: 9 samples were taken
- At termination of the administration period: 3 samples were taken
- Total samples: 12
The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date. The samples (1g potassium dicyanoaurate and 10mL vehicle) were shipped on dry ice via courier following advanced notice. The determinations were the responsibility of Allessa and are not covered by LPTs QAU program. The analyses were performed under GLP.
Duration of treatment / exposure:
Main study males (MS) once daily for up to 35 days (beginning 2 weeks prior to mating lasting up to one day before sacrifice until a minimum dosing period of 28 days was completed).
Main study females (MS) once daily, beginning 2 weeks prior to making and continuing up to, and including, day 3 post-partum of the day before sacrifice.
Toxicity females (TX) once daily, beginning on test day 1 and continuing up to, and including, test day 43.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Group 1: Control (vehicle)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
Group 2: 1 mg/kg b.w/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
Group 3: 3 mg/kg b.w/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
Group 4: 10 mg/kg b.w/day
Basis:
nominal in water
No. of animals per sex per dose:
96 animals (48 males and 48 females), 12 animals per sex and group, in addition to 20 females (5 animals per dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered in graduated doses (0, 1, 3 or 10 mg potassium dicyanoaurate/kg b.w/day) to 3 groups of males and females prior to, during and after mating to generate information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The dose levels were based upon the results of a dose-range-finding study in rats (LPT Study No. 30161). At the time of sacrifice or death, the animals were examined macroscopically. The adult animals were examined externally and internally. The reproductive organs and all the organs of the adult animals showing macroscopic lesions were preserved. The pups were examined externally.

DOSE-RANGING STUDY

In the dose-ranging study, male and female rats were treated with 0.1, 1.0 and 10 mg Potassium dicyanoaurate/kg b.w/day for 14 days. As no adverse signs had been observed until test day 14, the doses were increased to 20 and 30 mg b.w/day for the formerly low and intermediate dosed animals for the following 7 test days. The control animals and the formerly high dose animals (10 mg/kg) were also treated for 7 further test days.

Premature deaths occurred in one of the 5 females at 20 mg Potassium dicyanoaurate/kg b.w./day and in one of the 5 males and 3 of the 5 females at 30 mg Potassium dicyanoaurate/kg b.w/day within 3-7 days. Oral treatment with 20 or 30 mg Potassium dicyanoaurate/kg b.w/day caused pultaceous faeces, haemorrhagic nose/snout, reduced motility, pilo-erection, salivation, ptosis and/or pale eyes/ears and reductions in body weight and food consumption. The incidence of findings increased with the dose. Necropsy revealed test item-related changes in the gastrointestinal tract (stomach enlargement, haemorrhagic foci, reddened mucosa, ulcera) in one female treated with 10 mg/kg b.w/day and in nearly all male and female rats treated with 20 or 30 mg/kg b.w/day. In addition, an enlarged spleen was noted in several rats treated with 20 or 30 mg/kg b.w/day.

Furthermore, increases of the relative and absolute weights of the kidneys and stomach were recorded for the male and female rats treated with 10, 20 or 30 mg Potassium dicyanoaurate/kg b.w/day.

Positive control:
Not reported

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked regularly throughout the working day from 7.00 am to 3.45 pm. On Saturdays and Sundays animals were checked regularly from 7.00 am to 11.00 am, with a final check performed at approximately 3.30 pm.
- Records: Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
- Cage side 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
- Individual animals were observed before and after each dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment.
- Once before the first exposure and once a week thereafter, detailed clinical observations were made in five males from the study group and in all females of the toxicity assessment groups (five females/group).
- Clinical signs: changes in skin, fur, eyes, mucous membranes, occurrence of secretion and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes

FOOD EFFICIENCY: Yes
- 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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily by visual appraisal

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli), as well as the assessment of grip strength and motor activity assessment were conducted in males and females. The neurological screening was conducted two hours after dosing and before any blood sampling for laboratory examinations.
- Time schedule for examinations (5 randomly selected males per group): immediately prior to sacrifice on treatment day 35
- Time schedule for examination (all 5 females’ males per group): immediately prior to sacrifice on treatment day 44 (during lactation period on main study females)
- Dose groups that were examined: All dose groups
- Battery of functions tested: sensory activity (Gad 1982), grip strength (Meyer et al. 1979) and motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The male F0 animals (MS) were sacrificed on test day 35. Dams with offspring (MS) were sacrificed on day 4 post-partum. Females showing no signs of littering were sacrificed 24 days after the last day of the mating period. Animals for female toxicity assessment (TX) were sacrificed at the same time as the majority of the female main study animals (test day 44). Animals were sacrificed by cutting the aorta abdominalis under ether anaesthesia, exsanguinated, weighed, dissected and inspected macroscopically for any abnormalities or pathological changes. Organ weights were determined for 20 adult males and 20 females, including:
- Adrenal gland (2)
- Brain
- Heart
- Kidney
- Liver
- Spleen
- Thymus

HISTOPATHOLOGY: Yes
Histopathological examinations were performed on haematoxylin-eosin-stained paraffin sections of the following organs and tissues:
- Adrenal gland (2)
- Bone marrow (os femoris)
- Brain (cerebrum, cerebellum, brain stem)
- Epididymis (2)
- Gross lesions
- Heart (left and right ventricle, septum)
- Small intestine (duodenum, jejunum, ileum, incl. Peyer’s patches, Swiss roll method)
- Intestine, large (colon, rectum)
- Kidney and ureter (2)
- Liver
- Lungs (with mainstem bronchi and bronchioles, preserved by inflation)
- Lymph node (cervical)
- Lymph node (mesenteric)
- Mammary gland (females only)
- Nerve (sciatic)
- Ovary (2)
- Seminal vesicle
- Spinal cord (3 sections)
- Spleen
- Stomach
- Testicle (2)
- Thyroid (incl. parathyroid)
- Thymus
- Tissues masses or tumours (lymph nodes)
- Trachea (incl. larynx)
- Urinary bladder
- Uterus (incl. cervix and oviducts)
- Vagina
Statistics:
Analysis of normal distribution and homogeneity of variances was performed using the SHAPIRO-WILKS test and the BARTLETT test. Data not normally distributed or with heterogeneous variances between the groups were stepwise log- or rank- transformed.

One-way analysis of variance (ANOVA) was performed with non-transformed or log-transformed data. The KRUSKAL-WALLIS test was used for rank-transformed data. In case of significant differences (found by ANOVA or KRUSKAL-WALLIS test), intergroup comparisons with the control group were made by parametric or non-parametric DUNNETT multiple comparison tests (p ≤0.05 and p ≤0.01).

Statistical analyses of non-parametrical data were performed using the following settings:
Fishers exact test, n < 100 (p ≤0.05 and p ≤0.01)
Chi2 test, n ≤ 100 (p ≤0.05 and p ≤0.01)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
reduced motility, salivation
Mortality:
mortality observed, treatment-related
Description (incidence):
reduced motility, salivation
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No premature deaths were noted in the control group and in any of the other dose groups (1, 3 or 10 mg Potassium dicyanoaurate/kg b.w/day).
No changes in behaviour, the external appearance of the faeces were noted for the male animals of the low and intermediate dose group (1 or 3 mg test item/kg b.w/day) during the pre-mating, mating and post-mating period. Treatment with 10 mg test item/kg b.w/day caused a slight reduction in motility, slight salivation and increased urination.

No changes in behaviour, the external appearance or the faeces were noted for the female animals treated with 1 mg test item/kg b.w/day or the control animals during the pre-mating, mating period, gestation and lactation period. At 3 mg test item/kg b.w/day, increased urination was noted in 2 females.

No test-item related changes in external observations, body posture, movement and coordination or behaviour were noted for the evaluated male or female rats of the control group and the dose groups (1, 3 or 10 mg potassium dicyanoaurate/kg b.w/day).

BODY WEIGHT AND WEIGHT GAIN
MALES: No test item-related changes in body weight and body weight gain were noted for the male rats treated with 1 or 3 mg test item/kg b.w/day in comparison to the control group. The mean body weight of high dose males (10 mg test item/kg b.w/day) was marginally below the control values on days 22, 29 and 34, however, this was not deemed significant.

FEMALES: No test item-related changes in body weight and body weight gain were noted for the female toxicity rats of the treatment groups (1, 3 and 10 mg test item/kg b.w/day) in comparison to the control group. No test item-related differences in body weight and body weight gain were noted between the female rats of the control group and the female rats of the treatment groups (1, 3 and 10 mg test item/kg b.w/day) during the pre-mating period. A slight but statistically significant increase in body weight gain noted in the low dose group in the first test week (TD 1-8) showed no dose-response relationship and was considered not to be test item-related.

FOOD CONSUMPTION
No test item-related changes in food consumption were noted between the male and female rats treated with 1, 3 and 10 mg test item/kg b.w/day and controls during pre-mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No test item-related changes in the consumption of drinking water was noted for the male and female rats treated with 1, 3 and 10 mg test item/kg b.w/day by visual appraisal.

HAEMATOLOGY
MALES: No test item-related effects were noted for the haematological parameters of males in the low and intermediate dose groups (1 and 3 mg test item/kg b.w/day). However, in the high male dose group (10 mg test item/kg b.w/day) slight but statistically significant reductions were noted for the haemoglobin content (11.4%, p ≤0.05) and the number of erythrocytes (14.8%, p ≤0.01) in comparison to the control group. A statistically significant increase was noted for the reticulocyte count, in addition to a slight increase in the mean corpuscular volume (MCV), leading to a slight decrease for the mean corpuscular haemoglobin concentration (MCHC).

FEMALES: No test item-related influence was noted for the haematological parameters of the low and the intermediate female dose group (1 and 3 mg test item/kg b.w/day). However, comparable to the males, high dose females presented a slight but statistically significant reduction in haemoglobin content (11.4%, p ≤0.05) and for the number of erythrocytes (14.9%, p ≤0.01). Statistically significant increased values in comparison to the controls were noted for the reticulocyte count and mean corpuscular volume, leading to a slight decrease for the mean corpuscular haemoglobin concentration (MCHC).

CLINICAL BIOCHEMISTRY
MALES: No test item-related influences on the biochemical parameters were noted for the low dose group and for the intermediate dose group (1 and 3 mg test item/kg b.w/day). At the high dose level (10 mg test item/kg b.w/day) statistically significant reductions were ntoed for the albumin concentration (10.3%, p ≤0.01) for globulin concentration (18.7%, p ≤0.05) and accordingly for the total protein concentration (14.4%, p ≤0.01). Reductions in bilirubin, creatinine and calcium concentration were not considered to be test-item related.

FEMALES: No test item related influence was noted for the examined plasma or serum levels of the biochemical parameters in any of the treatment groups (1, 3 and 10 mg test item/kg b.w/day). Statistically significant decreases in globulin concentration and an increase in the albumin: globulin ratio, decreased chloride and creatinine concentration was not considered to be test item-related. Animals in the control group presented high variability and all individual values of the high dose group were within the LPT background data.

NEUROBEHAVIOUR
No adverse effects were noted in the evaluated male or female rats in any of the treatment groups (1, 3 or 10 mg test item/kg b.w/day) during observational screening or functional screening (grip strength and spontaneous motility).

ORGAN WEIGHTS
No differences in the body weight at autopsy was noted between the control group and treatment groups (1, 3 and 10 mg test item/kg b.w/day) for the main study male and female animals. No test item related differences were noted for the relative and absolute organ weights of males. No test item related differences between the control group and the low and intermediate dose group (1 and 3 mg test item/kg b.w/day) were noted for the absolute and relative organ weights. At the high dose level (10 mg test item/kg b.w/day) increases were noted in the absolute and relative organ weight of the liver and the spleen, which were considered to be test item-related.

GROSS PATHOLOGY
MALES: Necrospy revealed no test item-related changes during the macroscopic examination of the internal organs and tissues in the male rats treated with 1 mg test item/kg b.w/day. At 3 and 10 mg test item/kg b.w/day, gastric lesions of the forestomach, confirmed to be squamous cell hyperplasia (hyperkeratosis) and stomach erosions (haemorrhages) by microscopic examination, were reported. Multiple brownish-red foci on the left side of the thymus were noted in one male of the low dose group. This change was considered a spontaneous finding.

FEMALES: No test item-related changes were noted during the macroscopic examination of the internal organs and tissues in the female rats treated with 1 or 3 mg test item/kg b.w/day. At 10 mg test item/kg b.w/day, gastric lesions (forestomach) were noted in four females. These macroscopic changes were confirmed to be squamous cell hyperplasia (hyperkeratosis) by microscopic examination. Necropsy revealed a cystic left ovary in one high dose dam (filled with clear liquid) and in the non-pregnant control, these changes were considered to be spontaneous.

HISTOPATHOLOGY
MALES: No test item-related influence was noted on the qualitative stages of spermatogenesis. The histopathological examination performed on one testicle and one epididymis and histopathology of the interstitial testicular structure did not reveal any test item-related effects. Microscopic changes were noted for the forestomach (non-glandular mucosa: squamous cell hyperplasia/hyperkeratosis) in all animals at 3 and 10 mg test item/kg b.w/day and for the stomach erosions/haemorrhages. These lesions are considered to be test item-related.

FEMALES: Microscopic changes were noted for the forestomach (squamous cell hyperplasia/hyperkeratosis) for all females treated with 3 and 10 mg test item/kg b.w/day) and for the stomach erosions in all toxicity females treated with 10 mg test item/kg b.w/day. These lesions were associated with macroscopic findings and are considered to be test item-related.

Effect levels

Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No-observed-adverse effect level for systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Potassium dicyanoaurate demonstrated signs of systemic toxicity at 10 mg/kg b.w/day. The NOAEL was set at 3 mg Potassium dicyanoaurate/kg b.w/day.
Executive summary:

Conducted according the OECD Test Guideline 422, graduated doses of Potassium dicyanoaurate (0, 1, 3 or 10 mg /kg b.w/day) were administered to 3 groups of males and females to generate information concerning the systemic toxicity.

No premature deaths were noted. At 1 mg Potassium dicyanoaurate/kg b.w/day there was no evidence of systemic toxicity, no neurological effects and no treatment related differences in organ weights and macroscopic and microscopic pathology. Autopsy revealed gastric lesions at concentrations of Potassium dicyanoaurate >3 mg/kg b.w/day, which were confirmed by histopathological examination (squamous cell hyperplasia, hyperkeratosis, erosions, haemorrhages). These findings are considered to be local irritant effects and are not indicative of systemic toxicity. Increases in urination (during handling) was also noted in a few animals at 3 mg Potassium dicyanoaurate/kg b.w/day.

 

At the high dose group (10 mg Potassium dicyanoaurate/kg b.w/day) signs of systemic toxicity were noted in the form of reduced motility, slight or moderate salivation and increased urination. In addition, haematology investigations revealed changes in the erythrocyte parameters of the male and female rats. Clinical biochemistry revealed a decreased protein concentration in the plasma of the male rats. Changes in organ weights in the form of increased liver and spleen weights were noted in female rats. A tendency towards an increased spleen weight was noted in the male animals.

 

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 3 mg Potassium dicyanoaurate/kg b.w/day.