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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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 heigh
t 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 t
he drinking water or food. Samples of the drinking water are taken periodically by the Wasserwerk W
ankendorf 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
Vehicle:
water
Details on 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
Details on mating procedure:
- M/F ratio per cage: 1 male and 1 female per cage
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug or presence of sperm referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): 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. Female animals were placed in the animal room on the other side of the room with each dose group separated by an empty row..
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 a pplication 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
Details on study schedule:
Males: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 36 at maximum) until test day 47 (one day before sacrifice)
Females (with litter): 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 36 at maximum) until test day 64 to 71 (corresponding to lactation days 13 to 15).
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
- Rationale for animal assignment: random

Examinations

Parental animals: Observations and examinations:
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

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

Oestrous cyclicity (parental animals):
The oestrus cycle was monitored during the pre-mating and mating period from test day 15 (start of treatment) until one day before the first sign of mating was noted.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
Detailed histopathological examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of the interstitial testicular structure) of the selected males of groups 1 and 4 following H-E and PAS staining.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter. The litter size was adjusted by eliminating surplus pups following a randomization scheme.

PARAMETERS EXAMINED
As soon as possible after delivery, each litter was examined to establish the
number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the
presence of gross abnormalities. Any abnormal behaviour of the offspring would have been recorded.Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13. On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale. On PND4, blood samples for T4 hormone level determination were taken from 2 of the culled surplus pups. Nipples/areolae were counted in all male pups on PND 13 (shortly before scheduled sacrifice).

GROSS EXAMINATION OF DEAD PUPS:
Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
The thyroid of 1 male and 1 female pup from each litter was fixed in 7% formalin. Thyroid weight was determined after fixation.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: On test day 48 (after a dosing period of at least 4 weeks)
- Maternal animals: On lactation day 14 or 16

GROSS NECROPSY
The adult animals were examined macroscopically for any abnormalities or pathological changes.
All superficial tissues were examined visually and by palpation. brain, pituitary gland and cranial nerves were observed after the cranial roof was removed. After ventral midline incision and skin reflection all subcutaneous tissues were examined.
The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined.
The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.


HISTOPATHOLOGY / ORGAN WEIGHTS
The weight of the following organs was determined before fixation (where
applicable):
Adrenal gland (2), Spleen, Brain, Testicle (2), Epididymis (2), Thyroid, Heart, Thymus, Kidney (2), Liver, Ovary (2) with coagulating glands, Uterus including cervix
As a whole: prostate, seminal vesicles

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examination for gross abnormalities. The external reproductive genitals were examined for signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid of 1 male and 1 female pup from each litter was fixed in 7% formalin. Thyroid weight was determined after fixation.
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.
The statistical evaluations of the group indices (fertility index, gestation index, birth and live birth index, viability index and the pre- and post-implantation loss) using the number of corpora lutea, implantation sites and/or pups per group was done using StatXact 4.0.1 software, as such a calculation isnot possible in Provantis.
Reproductive indices:
The following indices were calculated for each group: male and female fertility indices, gestation index.
Offspring viability indices:
Birth index, live birth index, viablity index, post-implantation lost were determined for each litter and group.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 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 that was noted for the female animals at the high dose group. 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
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
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 parameters (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 not to be test item-related,
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No test item-related influence was noted on the mortality of pups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A reduced body weight was noted at the high dose level (450/375/300 mg test item/kg b.w./day) for the male and female pups on lactation day 13 (16.1 % below the value of the control group for the male and female pups combined, p ≤ 0.01).
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the thyroid weight on posnatal day 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The external macroscopic examination of the pups after sacrifice revealed no test item-related gross abnormalities.
Histopathological findings:
no effects observed
Description (incidence and severity):
The microscopic examination of the thyroids of the pups revealed no changes in the pups of the control group and the pups of the treatment groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No test item-related influence on prenatal development (pre- and post-implantation loss, number of pups born, number of stillbirths, birth and live birth indices) were noted at any of the tested dose levels.
During the postnatal development a reduced pup body weight was noted for the male and female pups of the high dose group 300 mg test item/kg b.w./day on lactation day 13.
No further adverse effects were noted on the postnatal development of the pups with respect to survival index, the endocrine/sexual development (T4 levels, anogenital distance, male nipples counting) and gross abnormalities.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
No test item-related influence was noted on the fertility and the gestation indices, the pre-coital time and the gestation length of F0 generation. The NOAEL for the reproductive parameters of the parental females was determined to be above 300 mg TCA/kg b.w./day, p.o.
A reduced pup body weight was noted for the male and female pups of the high dose group 300 mg test item/kg b.w./day on lactation day 13. The NOAEL based on postnatal development was established to be 150 mg TCA/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.

No test item-related influence was noted on the fertility and the gestation indices, the pre-coital time and the gestation length of F0 generation.

In F1 generation, no test item-related influence on prenatal development (pre- and post-implantation loss, number of pups born, number of stillbirths, birth and live birth indices) were noted at any of the tested dose levels. During the postnatal development a reduced pup body weight was noted for the male and female pups of the high dose group 300 mg test item/kg b.w./day on lactation day 13. No further adverse effects were noted on the postnatal development of the pups with respect to survival index, the endocrine/sexual development (T4 levels, anogenital distance, male nipples counting) and gross abnormalities. The NOAEL  for the reproductive parameters of the parental females was determined to be above 300 mg TCA/kg b.w./day, p.o. The NOAEL based on postnatal development was established to be 150 mg TCA/kg b.w./day, p.o..