Silver cyanide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2014 - 18 December 2015

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other: GLP-compliant, guideline study available as an unpublished report.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:

2015

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)

Test material

Test material information

Constituent 1

Reference substance name:

Silver cyanide

EC Number:

208-048-6

EC Name:

Silver cyanide

Cas Number:

506-64-9

Molecular formula:

CAgN

IUPAC Name:

silver(1+) cyanide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Silver cyanide
- Physical state: solid
- Stability under test conditions: Stable under test conditions
- Storage condition of test material: Ambient conditions (10 - 25°C in a tightly closed container)

Test animals

Species:

rat

Strain:

other: CD® Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 70 days
- Weight at study initiation: Males (MS): 295.7-332.1g; Females (MS): 198.4-238.2g; and Females (TX): 202.6-232.1 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)
- 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: 5 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

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: 1% aqueous hydroxypropyl methyl cellulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was supplied as a powder and suspended in the vehicle (1% aqueous hydroxypropyl methyl cellulose gel) to the appropriate concentrations for administration. The test item formulations were prepared once weekly and stored in a tightly closed container at room temperature (10 - 25°C) until use.

VEHICLE:
- Vehicle: Methocel solution (1% aqueous hydroxypropyl methyl cellulose gel)
- Lot/Batch No.: Batch no. 13D03-N03, FAGRON GmbH, 22885 Barbuttel, Germany
- Justification: Satisfactory stability for 7 days at room temperature was demonstrated for suspensions of 0.1 and 10 mg silver cyanide/mL in aqueous methocel solution (Allessa GmbH GLP Report No. B 079/2013).

Details on mating procedure:

Sexually mature male and female rats were randomly paired for monogamous mating:1 male and 1 female were placed in one cage during the dark period. The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug. If findings were negative, mating was repeated. The day of conception (day 0 of gestation) was considered to be the day on which sperm was found. This procedure was repeated until at least 8 pregnant dams were available for each group. The prematurely deceased male was replaced by another male of the same treatment group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations of silver cyanide-vehicle mixtures were analysed by Allessa GmbH for homogeneity and concentration. Immediately after preparation of test solutions, samples were taken from the top, middle and bottom of the container. 3 samples per dose level per group were conducted per week (9 samples per week). The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date.

Duration of treatment / exposure:

MAIN STUDY MALES (MS): Once daily. Beginning 2 week prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed. In detail, the male rats were dosed from test day one until, and including, test day 38 and were sacrificed on test day 39.

MAIN STUDY FEMALES (MS): Once daily. Beginning 2 weeks prior to mating and continuing up to and including, day 2 post-partum or the day before sacrifice. In detail, the female rats were dosed between test day one and test day 40 (first sacrificed female on test day 41) or test day 54 (last sacrificed female on test day 55).

TOXICITY FEMALES (TX): Once daily. Beginning on test day 1 and continuing up to, and including, test day 39. The animals were sacrificed on test day 40.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5 mg Silver cyanide/kg b.w/day
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
15 mg Silver cyanide/kg b.w/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
50/40 mg Silver cyanide/kg b.w/day
Basis:
nominal conc.

No. of animals per sex per dose:

In the main study, 96 animals (48 males, 48 females), 12 animals per sex and group. In addition, 20 females (5 females per group) were used fir for toxicity assessment.

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered in graduated doses (0, 5, 15 or 50/40 mg silver cyanide/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 an acute toxicity study (LPT Study No. 30163) and a 14-day dose ranging study (LPT Study No. 30164). The acute toxicity study showed that a dose level of 55 mg Silver cyanide /kg b.w was in excess of a maximum tolerated dose due to the severity of the clinical observations (i.e. reduced motility, ataxia, tremor, reduced muscle tone, dyspnoea, convulsions). In the dose-ranging study, rats were treated with 20, 30-50 and 40 mg Silver cyanide/kg b.w/day for 14 days. The intermediate dose of 30 was enhanced to 50 mg Silver cyanide/kg b.w/day after 14 days. At 50 mg Silver cyanide/kg b.w/day, signs of systemic toxicity were noted in the form of salivation, piloerection and/or reduced motility for the male and female rats. The male rats additionally showed a reduction in body weight and body weight gain.

The intermediate and the low doses proposed give an approximate three-fold separation of doses to allow the dose response for any effect at the high dose to be determined. The results of the dose-ranging study indicated that the dose response for Silver cyanide via the oral route is steep.

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.

Positive control:

None reported

Examinations

Parental animals: Observations and examinations:

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, grip strength and motor activity

Oestrous cyclicity (parental animals):

Not reported

Sperm parameters (parental animals):

Detailed histopathological examination was performed on one testicle and one epididymis with special emphasis of the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of interstitial testicular structure of the selected male animals on the main study groups 1 and 4 following haematoxylin-eosin and PAS staining.

Litter observations:

The following parameters were evaluated:
- Number of pregnant females
- Duration of pre-coital time
- Gestation length*
* The length of gestation was calculated from day 0 of pregnancy (gestation day), until one day before lactation day (day without any signs of littering in the morning) and consistency including the day(s) of parturition.

Copora lutea
- Number per dam
- Absolute number per group
- Mean per group

Implantation sites
- Number per dam
- Distribution in the uterine horns
- Absolute number per group
- Mean per group

Live pups were counted and sexed and litters weighted. Any abnormal behaviour of the offspring was recorded.
- Number of pups absolute (at birth, 4-days post partum)
- Number of pups per dam (at birth, 4-days post partum)
- Number of stillbirths (absolute and per dam)
- Number of pups with malformations (absolute and per dam)

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
The male F0 animals (MS) were sacrificed on test day 39 (with the exception of the prematurely deceased animal no. 75). 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 40). 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

Postmortem examinations (offspring):

MALFORMATIONS
Dead pups and pups sacrificed at day 4 post-partum were carefully examined externally for gross abnormalities. Malformations are abnormalities that are considered to have a significant adverse effects on the foetus with or without fatal consequence (EPA 1992). They are persistent structural or functional deviations outside the normal biological variation. Hence, malformations are morphological and/or functional damages, which if occurring in humans, would endanger the existence or social position. Malformations monitored included:

- Acephaly
- Acrania
- Cranioschisis
- Encephalocele
- Exencephaly
- Agnathia
- Brachygnathia
- Cleft lip
- Cleft palate
- Anal atresia
- Omphalocele
- Spina bifida
- Adactyly
- Brachydactyly
- Oligodactyly
- Polydactyly
- Acaudia
- Brachycaudia
- Rudimentary
- Agenesis
- Misalignment
- Bone fusion
- Supernumerary

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 nonparametric DUNNETT multiple comparison tests (p ≤ 0.05 and p ≤ 0.01).
Statistical analyses of non-parametrical data (i.e. the reproductive indices) were performed using the following settings:
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)

Reproductive indices:

The following indices were calculated for each group:
Male fertility Index [%] = (No. males with confirmed female insemination / No. rats) x 100
Female fertility Index [%] = (No. pregnant rats / No. rats) x 100

The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed litters.

Gestation Index [%] = (No. dams with live pups / No. pregnant rats) x 100

Offspring viability indices:

For each litter and group, the following indices were determined:
Birth Index [%] = (Total no. of pups born / Number of implantation sites) x 100
Live Birth Index [%] = (No. pups alive on day 0-1 of lactation / Total no. pups) x 100
Survival Index [%] = (Number of pups alive on day 4 / Number of pups alive on day 0-1) x 100
Pre-implantation loss [%] = (Corpora lutea - Implntations / corpora lutea) x 100
Post-implantation loss [%] = (Implantations - Living foetuses / Implantations) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

high dose group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

high dose group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

high dose group

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

high dose group

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
No premature deaths were noted in the control group and in the low or intermediate dose group (5 or 15 mg Silver cyanide/kg b.w/day). After oral treatment with the high dose (50 mg Silver cyanide/kg b.w/day) one male and two female rats were found dead. A further high dose female treated with 40 mg Silver cyanide/kg b.w/day was found dead on test day 21 (i.e. 10 days after dose reduction). Reduced motility and salivation were observed prior to death and all premature deaths are considered as test item-related.

Pre-lethal symptoms were noted in the form of extremely reduced motility and/or extreme salivation. Macroscopic inspection during dissection revealed lesions in the stomach, the lungs and the brain/skullcap in one male. Residual test item was noted in the stomach of females animals, as death occurred before the absorption of the administered test item formulation.

At 50/40 mg Silver cyanide/kg b.w/day reduced motility and piloerection were noted for all male and female animals at the beginning of the study between test days 1 to 4. Four of the 12 males (MS) and 14 of 17 females (MS/TX) revealed prone position on 1 or 3 days between test days 2 and 3. Salivation was noted on a few to several test days in all surviving male and female animals, mainly starting on test day 15 and 16. Tremors, an opisthotonus, laboured breathing and an increased respiratory rate were additionally noted in a few of the surviving animals on 1 or 2 test days.

No details clinical observations were noted in MS males on test day 37 and females on test day 39.

BODY WEIGHT AND WEIGHT GAIN
MALES: At 15 and 50/40 mg Silver cyanide/kg b.w/day slight delays in body weight gain were noted in the male animals during the first 2 test weeks. This led to a body weight that was between 3.7% (min) and 5.3% (max) below the control group between test days 15 and 38 for the rats of the intermediate dose group (statistically not significant). For the rats of the high dose group the body weights between test days 15 and 38 were reduced by 5.0 - 7.9% in comparison to the control group (statistically significant on TD 36 at p ≤ 0.05). Body weight gain for the whole study period (from TD 1 until TD 38) was statistically significantly reduced for the rats dosed with 15 and 50/40 mg Silver cyanide/kg b.w/day (p≤0.05) in comparison to the control group.

FEMALES: No test item-related changes in body weight and body weight gain were noted in any of the females.

FOOD CONSUMPTION
No test item-related changes in food consumption were noted.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No test item-related changes in food consumption were noted.

HAEMATOLOGY
No adverse test item-related changes were noted.

CLINICAL BIOCHEMISTRY
At 50/40 mg Silver cyanide/kg b.w/day a statistically significant increase (p ≤0.01) was noted for the chloride concentration on test day 15. No adverse test item-related changes were noted in females.

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 grip strength. A tendency towards a reduced spontaneous motility was noted in the female animals at the 50/40 mg Silver cyanide/kg b.w/day.

ORGAN WEIGHTS
MALES: No test item-related changes in organ weight were noted in any of the males.

FEMALES: At 50/40 mg Silver cyanide/kg b.w/day increased organ weights (p ≤0.05, p ≤0.01 or not significant) were noted for the relative and the absolute organ weights of the heart (+51% and +60% for absolute and relative weights, respectively), left kidney (+14%, +21%), right kidney (+14, +20%) and the liver (+25%, +34%).

GROSS PATHOLOGY
No test item-related changes were noted in the post-mortem.

HISTOPATHOLOGY
MALES: No test item-related changes were noted for the examined organs and tissues from 5 selected male rats of the high dose group. A detailed examination of the qualitative stages of spermatogenesis and the interstitial testicular structure revealed no test item-related changes.

FEMALES: At 50/40 mg Silver cyanide/kg b.w/day, test item related microscopic changes were noted in the form of liver and/or lungs with congestion.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No test item-related differences were noted between the survival index of the pups from the dams of the control group and the pups from the dams treated with 5 or 15 or 50/40 mg Silver cyanide/kg b.w/day.

BODY WEIGHT (OFFSPRING)
No test item-related difference was noted between the mean body weight of a pup from the dams of the control group and the mean body weight of a pup from the dams treated with 5, 15 or 50/40 mg Silver cyanide/kg b.w/day on lactation days 1 and 4. Slightly lower body weight was noted for the male and female pups of the intermediate group on lactation day 1 and 4 and two runts were noted, however, these sporadic occurrences are not considered to be test item-related.

The total litter weights from the dams of the control group and those from the treatment groups (5, 15 and 40/50 mg Silver cyanide/kg b.w/day) revealed no test item related differences on lacataion day 1 and 4.

GROSS PATHOLOGY (OFFSPRING)
No gross abnormalities (e.g. malformations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 5, 15 or 50/40 mg Silver cyanide/kg b.w/day after sacrifice on lactation day 4.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted on the development of the pups (F1-Generation) with respect to survival index, body weight, gross abnormalities

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No adverse effects on fertility and reproductive parameters were identified, the NOAEL was >50/40 mg Silver cyanide/kg b.w/day.
No adverse effects on the development of pups were identified, the NOAEL was >50/40 mg Silver cyanide/kg b.w/day.

Executive summary:

The test item was administered in graduated doses (0, 5, 15 or 50-40 mg silver cyanide/kg b.w/day) to 3 groups of males and females to obtain information on possible effects of the test item on reproductive toxicity, fertility and pup development, according to OECD 422 guideline study design. The high dose level was reduced as of test day 12 due to mortality and clinical signs.

 

The administration of the main study rats started two weeks before mating on day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 39 for all parental male rats, lactation day 4 for all parental females and on test day 40 for the toxicity assessment of non-mated females.

 

Analysis performed by Allessa GmbH confirmed that the suspensions of Silver cyanide were correctly prepared and generally within acceptable limits (80 -120%). The concentrations of Silver cyanide in the test item vehicle formulations were between 81.2% and 107.8% of the nominal concentration in the high dose group, between 38.2% and 94.7% of the nominal concentration in the intermediate dose group and between 40.7% and 107.4% of the nominal concentration in the low dose group.

Whilst indications of general toxicity were observed at 50/40 mg Silver cyanide/kg b.w/day, no influence was noted on the fertility and the reproductive parameters of the parental generation (F0-generation) with respect to fertility, pre-coital time, gestation length, gestation index, number of stillbirths, birth and live birth index and the pre- and post- implantation loss at any of the examined dose levels. No adverse effects were noted on the development of the pups (F1-generation) with respect to survival index, body weight or gross abnormalities.

No adverse effects on fertility and reproductive parameters were identified, the NOAEL was >50/40 mg Silver cyanide/kg b.w/day.

No adverse effects on the development of pups were identified, the NOAEL was >50/40 mg Silver cyanide/kg b.w/day.