Potassium cyanate

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2021 to October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

15 July 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental animals: 10 weeks as requested by the agency
- Basis for dose level selection: The dose setting is based on findings obtained from previous studies with Potassium Cyanate in rats , in particular the Reproduction/Developmental Toxicity Screening Test with Potassium Cyanate in the Rat
- Exclusion of extension of Cohort 1B
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
- Exclusion of developmental immunotoxicity Cohort 3
- Route of administration: oral (gavage)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium cyanate

EC Number:

209-676-3

EC Name:

Potassium cyanate

Cas Number:

590-28-3

Molecular formula:

KOCN

IUPAC Name:

potassium cyanate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot number of test material: A4H0801

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for toxicity and reproduction toxicity studies and the test guidelines were designed to use the rat.
The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity and reproduction toxicity studies and well-known fertility parameters.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P0 males: 6 weeks; P0 Females: 5 weeks
- Weight at study initiation: P0 Males: 173-199 g; Females: 99-127 g;
- Fasting period before study: No
- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Mated females were housed individually.
Males after mating: 2 animals/cage
F1 offspring (after weaning): Not more than 3 animals of the same sex/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: May 18th, 2021 To: December 20th, 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of the Test Facility not longer than for three days before the use and were stored at room temperature. Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and measured on five occasions. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.

VEHICLE
- Concentration in vehicle: 4, 12 and 24 mg/mL
- Amount of vehicle: 5 mL

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: The mating period was prolonged for some female animals in high dose group by a week to ensure the appropriate number of litters by changing of mating partners.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test item in the dosing formulations used for animal’s treatment was checked five times during the study. Concentration of Potassium Cyanate in the dosing formulations varied in the acceptable range between 94 % and 103 % of the nominal values and formulations were homogenous thereby confirming the proper dosing of parental animals (P) and F1 generations.
The suitability of the chosen vehicle – distilled water – for the test item at the intended concentrations was analytically verified up front. A sufficient recovery, stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery from distilled water was 99 and 94.4 % of nominal concentrations at 2 mg/mL and 50 mg/mL, respectively. Potassium Cyanate was stable at 2 mg/mL and 50 mg/mL concentrations for three days at room temperature.

Duration of treatment / exposure:

Overall treatment period was 127-129 days for male parental animals.
Overall treatment period was up to 114-135 days for female parental animals.
Non-pregnant and not delivered females were administered for 105 or 114 days and moribund animal for 43 days.
F1 offspring in Cohort 1A were administered up to post-natal day 91-111 (approximately 10-13 weeks).
F1 animals of Cohort 1B were administered up to post-natal day 98-114 (approximately 11-16 weeks).

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

120 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

26 P0 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose setting is based on findings obtained from previous studies with Potassium Cyanate in rats (Reproduction/Developmental Toxicity Screening Test with Potassium Cyanate in the Rat, study no. 953-421-5039) and in agreement with the Sponsor. In this study, Potassium Cyanate caused clinical signs (male and female), severely depressed body weight development and food consumption (male and female), macroscopic and histopathology changes in stomach (male), lowered organ weight (epididymides, seminal vesicle with coagulating gland and prostate as a whole) in Han:WIST rats at 250 mg/kg bw/day by oral gavage as investigated in this study. There were no test item related adverse effects at 20 or 100 mg/kg bw/day. Potassium Cyanate reduced the reproductive performance, impaired the impregnation rate in parental animals at 250 mg/kg bw/day (fertility and gestation indices, duration of pregnancy, number of implantation sites, number of births). The development of the F1 offspring was adversely affected (body weight) from birth to post-natal day 21 after repeated oral administration of dams at 250 mg/kg bw/day. 100 mg/kg bw/day (female) and 250 mg/kg bw/day (male and female) caused reduced body weight and food consumption in F1 animals during the two weeks treatment period after weaning.
Potassium Cyanate showed rapid absorption proving exposure towards the test item and elimination in rat’s plasma and there was no significant difference between sexes. Further, plasma data after repeated administration indicate that dose levels applied did not lead to saturation of toxicokinetic processes.
The high dose was selected based on the clear systemic effect at 250 mg/kg bw/day in the OECD 421 study, and the expectation that due to the prolonged pre-mating period compared to the preliminary study effects on reproduction might occur already at lower dose levels. The high dose level should induce toxic effects but no mortality or severe suffering of animals. The mid dose level is expected to produce minimal to moderate toxic effects. The low dose should produce no observable indications of toxicity.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Observations: Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- Observations: skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
Parental (P) males were weighed on the first day of dosing (Day 0), weekly thereafter and on the day of necropsy.
Parental (P) females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum days 0 (within 24 hours after parturition), 4, 7, 14 and 21.
F1 animals selected for follow-up examinations were weighed on post-natal day 22, then twice a week until post-natal day 42 and once weekly thereafter.
For selected F1 offspring, the body weight was recorded on the day when they attained puberty – completion of balano-preputial separation or vaginal patency.
Fasted body weight was measured on the day of necropsy for all animals (P and F1).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

URINALYSIS
The following parameters were evaluated in 10 selected test animals per sex per dose of P and F1 Cohort 1A generation:
Nitrite (NIT), pH, Glucose (GLUC), Urobilinogen (UBG), Bilirubin (BIL), Ketone (KET), Blood, Leucocytes (LEU), Specific Gravity (SG), Protein (PROT), Volume (VOL), Sediment (SED), Colour, Clarity

HEMATOLOGY/BLOOD COAGULATION
The following parameters were measured in 10 selected animals per sex per dose of P and F1 Cohort 1A generation:
White blood cell (leukocyte) count (WBC), Red blood cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) hemoglobin (MCH), Mean Corpuscular (erythrocyte) hemoglobin concentration (MCHC), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count, Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT).

CLINICAL CHEMISTRY
The following parameters were measured in 10 selected animals per sex per dose of P and F1 Cohort 1A generation:
Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Urea concentration (UREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Sodium concentration (Na+), Potassium concentration (K+), Albumin concentration (ALB), Total protein concentration (TPROT)

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4, TSH) as follows:
- from 10 parent male animals/sex/ group at termination;
- from surplus pups at PND 4 (pooled by litters)
- from F1 pups not selected for cohorts on PND 22
- from 10 P dams on post-partum day 22;
- from 10 adult F1 Cohort 1A male and female animals/group at termination;

SEXUAL MATURITY
Sexual maturity of selected F1 animals (Cohort 1A and Cohort 1B) was examined by observing balano-preputial separation on postnatal day 25 or vaginal patency (between postnatal days 25 and 40). The body weight was determined on the day when balano-preputial separation or vaginal patency was completed. Additionally, the day of appearance of first cornified vaginal smear was monitored after vaginal patency and the time interval between these events was determined in female animals in Cohort 1A.

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears from each parental (P) female animal daily for two weeks before the mating started (i.e., after 8 weeks of pre-mating treatment) and daily for two weeks before the necropsy for F1 Cohort 1A (PND79-92) and Cohort 1B (PND86-99) female animals.
Vaginal smears were also prepared and estrous cycle was monitored daily during the mating period until evidence of copulation in parental animals (P) and on the day of the necropsy of all female animals (P and F1 Cohort 1A and Cohort 1B).
Vaginal smears were examined for all F1 Cohort 1A females selected for follow-up examinations after the onset of vaginal patency until the first cornified smear was recorded thus determining the time interval between these events.

Sperm parameters (parental animals):

Sperm parameters were measured in all male animals in P generation and in F1 generation in Cohort 1A.
The one-side testes and epididymides were used for examinations. The weights of one-side testes and epididymides were determined and recorded.
Sperm from the ductus deferens was collected for evaluation of sperm motility and morphology at the necropsy. Both numbers of motile and immotile sperms were recorded. Two samples were prepared from each animal. For the determination of the sperm motility, the mean percentage of motile sperms was determined.
A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails). The total number of sperms in homogenization of testis was enumerated.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups on PND13.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after weaning of offspring.
- Maternal animals: All surviving animals at least up to and including PND 21.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively, for high dose and control animals:
Organ weights (all parental (P) animal and all adult F1 animals of Cohort 1A):
- Uterus (with oviducts and cervix)
- Ovaries
- Testes
- Epididymides
- Prostate (dorsolateral and ventral parts combined)
- Seminal vesicles with coagulating glands as one units (with their fluids)
- Brain
- Liver
- Kidneys
- Heart
- Spleen
- Thymus
- Pituitary
- Thyroid + parathyroid glands (post-fixation)
- Adrenal glands

List of organs preserved and examined microscopically (all parental (P) animal and all adult F1 animals of Cohort 1A):
- Adrenal glands
- Bone with marrow and joint (femur)
- Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
- Eyes (lachrymal gland with Harderian glands and optic nerve)
- Mammary gland (male and female)
- Heart
- Kidneys
- Large intestines (caecum, colon, rectum),
- Liver
- Lungs (with main stem bronchi; inflation with fixative and then immersion;)
- Lymph nodes (submandibular, mesenteric; popliteal for Cohort 1 animals)
- Muscle (quadriceps)
- Esophagus
- Pancreas
- Pituitary
- Salivary glands (submandibular)
- Sciatic nerve
- Sexual organs (testes, epididymides, seminal vesicle with coagulating gland, prostate ovaries, uterus with oviduct, vagina)
- Small intestines (duodenum, ileum, jejunum including Peyer’s patches)
- Spinal cord (at three levels: cervical, mid-thoracic and lumbar)
- Spleen
- Stomach
- Thymus
- Thyroid + parathyroid
- Trachea
- Urinary bladder

In animals of Cohort 1B, the weight of following organs were determined:
- Uterus (with oviducts and cervix)
- Ovaries
- Testes
- Epididymides
- Prostate (dorsolateral and ventral parts combined)
- Seminal vesicles with coagulating glands as one unit (with their fluids)
- Brain
- Pituitary
In adult F1 animals in Cohort 1B, the uterus (with oviducts and cervix), ovaries, testes, epididymides, prostate (dorsolateral and ventral parts combined), seminal vesicles with coagulating glands as one unit (with their fluids), brain and pituitary were processed up to block stage for later examinations if necessary.

OVARIAN FOLLICLE COUNTS
In the adult female animals of Cohort 1A (control and high dose groups) a quantitative evaluation of ovaries was performed. At quantitative histopathological examination, the number of primordial, primary and small growing (secondary and tertiary) follicles, as well as corpora lutea was determined in ovary sections stained with hematoxylin and eosin by light microscopy in all female animals in the control and high dose group (n=20/ group). No further follicular enumeration was considered necessary in the low and mid dose treated female animals as there were no test item related changes at the high dose (high dose group counts met well the control values).
Corpora lutea assessment was conducted in parallel with estrous cyclicity testing. Oviduct, uterus and vagina were examined for appropriate organ-typic development.

SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS
At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) as follows:
- weighing of the lymph nodes associated with and distant from the route of exposure (submandibular and popliteal lymph nodes);
- splenic lymphocyte subpopulation analysis: CD4+ (Helper T cells) and CD8+ (Cytotoxic T cells) T lymphocytes, B lymphocytes and natural killer (NK) cells were identified by a validated flow cytometry method. Immuno-staining and immunophenotyping of spleen lymphocytes were carried out after preparation of single cell suspensions from one half of each provided spleen. For further information please refer to the attached background material.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations macroscopic and microscopic examination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGTHS
For 10 male and 10 female F1 pups per group, not selected for Cohorts – from as many litters as possible – brain, spleen and thymus were weighed and preserved in 4 % buffered formaldehyde solution. In addition mammary tissues were preserved in the same solution.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant results at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
For the comparison of two groups, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
Frequency of toxic response, pathological and histopathological findings by sex and dose were calculated.
Results were evaluated in comparison with values of control group (i.e. control value).

Reproductive indices:

Copulatory Index (Measure of animals ability to mate):
Males: Number of males with confirmed mating / Total number of males cohabited x 100
Females: Number of sperm positive females / Total number of females cohabited x 100

Fertility Index (Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant):
Males: Number of males impregnating a females / Total number of males with confirmed mating x 100
Females: Number of pregnant females / Number of sperm positive females x 100

Gestation Index (Measure of pregnancy that provides at least one live pup):
Number of females with live born pups / Number of pregnant females x 100

Offspring viability indices:

Formulas for Calculation of Pup Mortality and Sex Ratio Indices:
Post-implantation mortality: Number of implantations – Number of liveborns / Number of implantation x 100
Post-natal mortality: Number of liveborns – Number of live pups on PND 13 / Number of liveborns x 100
Survival Index: Number of live pups on PND 13 / Number of liveborns x 100
Sex ratio: Number of pups examined – Number of pups males (females) / Number of pups examined x 100

Normalized anogenital distance: Absolute anogenital distance/(body weight)^1/3

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Adverse clinical signs related to the test item were not detected at the clinical observations.
Species specific signs were only found in two animals: Reddish colored hairs around the left eye (1/26 male at 20 mg/kg bw/day, between Days 14 and 126) and scar on the right-side scapula (1/26 female control (1/2 non-pregnant) between Days 56 and 95).
Scar on the skin and reddish colored hairs around the eye (sign of enhanced production of porphyrin) are species-specific finding, which are also observed in untreated experimental rats of this strain with similar age. These were individual findings with low incidence and were thus not considered related to the treatment.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in male and female animals in control, 20, 60 or 120 mg/kg bw/day groups.
One female animal was early euthanized at 120 mg/kg bw/day (1/26) on Day 43. Clinical observations were noted for this female animal during the two weeks before euthanasia (body weight loss from Day 28, clinical signs – decreased activity, decreased body tone, piloerection – between Days 35 and 43). Moribund state was identified and animal was subjected to euthanasia and to a complete necropsy on Day 43. Based on necropsy and histopathological findings, mis-swallowing caused the death of this female animal and it could be considered as individual disease leading to the moribund state.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was statistically significantly depressed in parental male animals at 60 and 120 mg/kg bw/day from Days 14 and 7, respectively, up to the end of the study and in female animals administered with 120 mg/kg bw/day from Day 28 and during the gestation and lactation period.
Slight changes in the mean body weight of male animals at 60 mg/kg bw/day and in female animals at 120 mg/kg bw/day during the gestation period were judged to be toxicologically irrelevant as the difference with respect to the control remained less than 10 %.
The mean body weight was comparable with their control in male animals at 20 mg/kg bw/day and in female animals at 20 and 60 mg/kg bw/day during the entire observation period.
A lower mean body weight was observed when compared to the control in male animals at 60 mg/kg bw/day (between -3 and -8 %) from Day 14 and at 120 mg/kg bw/day (between -3 and -18 %) from Day 7 up to the end of the treatment period.
Statistical significance with respect to the control was found at the lower mean body weight of female animals at 120 mg/kg bw/day from Day 28 and during the gestation and lactation periods (between -3 and -8 % respective to control).
The mean body weight gain of male animals remained below the control value in the most cases and the difference with respect to the control was statistically significant on several days as follows:
- at 20 mg/kg bw/day: between Days 28-35
- at 60 mg/kg bw/day: from Day 0 up to Day 49 by weekly interval, between Days 69-76, 90-97 and if summarized between Days 0 and 125
- at 120 mg/kg bw/day: between Days 0-69 by weekly interval, between Days 76-83, 83-90, 118-125 and between Days 0 and 125 (summarized)
The summarized body weight gain of male animals was dose dependently lower than in the control group at 60 and 120 mg/kg bw/day (-12 and -30 %, respectively).
In the female animals, the mean body weight was mostly comparable to the control at 20 and 60 mg/kg bw/day during the pre-mating, mating and lactation periods.
Some sporadic statistically significant difference with respect to the control were detected at lower or higher mean body weight gain as follows:
- 20 mg/kg bw/day: between Days 21-28, 28-35, between gestation days 7-14, 0-21 and between lactation days 14-21
- 60 mg/kg bw/day: between Days 21-28 and 63-69
- 120 mg/kg bw/day: between Days 21-28, 35-42, 42-49, 56-63 and 0-69, between gestation day 14-21 and 0-21, between lactation day 7-14
The summarized mean body weight gain was lower than in the control in female animals at 120 mg/kg bw/day during the pre-mating period (-15 %), gestation (-11%) and lactation (-11 %).
The slightly reduced mean body weight gain resulted in only minor changes in the mean body weight in male animals at 20 and 60 mg/kg bw/day and in female animals at 20, 60 and 120 mg/kg bw/day in each dose group (8 % or less relative to control even at 120 mg/kg bw/day).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was reduced in parental male animals at 60 and 120 mg/kg bw/day and in female animals at 120 mg/kg bw/day consistently during the entire treatment period. The reduction was dose related in male animals and was in full accordance with body weight development in both sexes.
In the male animals at 20 mg/kg bw/day, the mean daily food consumption was similar to their control (pre-mating and post-mating periods).
At 60 mg/kg bw/day, statistical significance with respect to the control was detected at the lower mean daily food consumption of parental male animals from the second week up to the termination of the study.
The mean daily food consumption was lowered in male animals at 120 mg/kg bw/day from week 1 up to and including week 18 (termination).
The mean daily food consumption was similar to the control in female animals at 20 mg/kg bw/day during the pre-mating, gestation and lactation periods except for week 3, when the mean daily food consumption significantly exceeded the control.
At 60 mg/kg bw/day, the mean daily food consumption was lowered with respect to their control on weeks 2, 4, 6, 7, 8, 9 and on gestation week 1.
In the parental female animals, the mean daily food consumption was statistically significantly lower at 120 mg/kg bw/day from week 2 up to week 10, during gestation and lactation period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse changes in the examined hematological parameters in parental male or female animals at 20, 60 or 120 mg/kg bw/day.
In the male animals, statistical significance was detected at the slightly lower mean percentage of eosinophil granulocytes (EOS) at 20 mg/kg bw/day, at the lower mean white blood cell count (WBC), higher mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH), at higher mean percentage of reticulocytes (RET) and shorter mean prothrombin time (PT) at 60 mg/kg bw/day when compared to the control.
At 120 mg/kg bw/day, a lower mean corpuscular volume and higher mean corpuscular hemoglobin concentration were observed comparing to their control.
In the female animals at 20 mg/kg bw/day, all the examined hematological and blood coagulation parameters were comparable to the control.
Statistical significance with respect to the control was detected at the slightly higher mean percentage of neutrophil granulocytes (NEU) along with lowered mean percentage of lymphocytes (LYM), at higher mean corpuscular volume, mean corpuscular hemoglobin content and at higher mean percentage of reticulocytes in the female animals at 60 mg/kg bw/day.
In the female animals at 120 mg/kg bw/day, lowered mean white blood cell count, lower mean red blood cell count (RBC), higher mean corpuscular volume and mean corpuscular hemoglobin content were observed when compared to their control.
The individual values of the above-mentioned parameters (WBC, EOS, MCV, MCH, RET, PT) met well the historical control (i.e., values were within or close to the ranges) in male and female animals, where relevant. There were no changes in related hematological parameters. Therefore, the differences in these parameters were considered to have little or no toxicological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examined clinical chemistry parameters were not adversely affected in parental male or female animals at 20, 60 or 120 mg/kg bw/day.
In the male animals at 20 mg/kg bw/day, statistical significance was detected at the slightly lower mean concentration of total bilirubin (TBIL), glucose (GLUC) and total protein (TPROT) when compared to the control.
At 60 mg/kg bw/day, statistical significance with respect to the control was detected at the lowered mean total bilirubin and total protein concentrations.

At 120 mg/kg bw/day, lowered mean activity of alanine aminotransferase (ALT), lowered mean concentrations of total bilirubin, creatinine (CREA), glucose, albumin (ALB), total protein and elevated mean concentration of potassium (K+) were observed when compared to the control.
In the female animals, the examined clinical chemistry parameters were predominantly comparable with the control at 20, 60 and 120 mg/kg bw/day. Statistical significance with respect to the control was only detected at higher mean activity of aspartate aminotransferase (AST) at 60 mg/kg bw/day.
Toxicological relevance of these changes is questionable due to the minor degree (values were within or close to normal ranges) or the lack of related findings (clinical and organ pathology).

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The thyroid hormone (FT3, FT4 and TSH) levels were not affected by the test item in parental male and female animals (20, 60 and 120 mg/kg bw/day) and in offspring sampled on post-natal day 22.
Slight, statistically significant difference with respect to their control was detected at the lower mean FT4 level in parental male animals and lower mean TSH level in female animals at 120 mg/kg bw/day.
The FT3, FT4 and TSH levels were comparable in PND22 offspring in the control, 20, 60 and 120 mg/kg bw/day groups.
The mentioned slight changes FT4 and TSH levels in parental animals were judged to be variations and of no toxicological relevance as values were well within the normal physiological ranges (historical control). Moreover, the weight and cell morphology of thyroid glands were similar to their control in parental animals.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examined urine parameters were comparable in the parental male and female animals in the control and 20, 60 and 120 mg/kg bw/day groups.
Urinalysis revealed statistically significantly lower mean volume of urine in female animals at 120 mg/kg bw/day .
The sediment was positive in one female animal at 60 mg/kg bw/day (1/10) due to the presence of leucocytes (LEU).
The difference with respect to the control were with minor degree and in the lack of related findings the change in volume was considered to be toxicologically not relevant.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Potassium Cyanate did not cause any toxic or other test item related lesions detectable by histological examinations of the investigated organs of experimental male and female animals.
In early euthanized female animal at 120 mg/kg bw/day, histological examination revealed acute purulent pneumonia in the lungs and acute purulent epicarditis in the heart as cause of death. No toxic lesions (degeneration, necrosis) were detected in the other investigated organs.
The acute pneumonia and epicarditis could be in possible connection with the treatment procedure, and could be considered as individual disease.

Terminal necropsy
The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in all parental male animals in the control and 120 mg/kg bw/day groups.
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphology in the testes of investigated control and treated animals. The histological picture of epididymides was normal.
In the parental female animals at 120 mg/kg bw/day and control groups, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
In the uterus, endometritis was observed in single female animals at 60 mg/kg bw/day (1/4) and at 120 mg/kg bw/day (1/25).
Dilatation of uterine horns was detected in several female animals (2/26, 6/6, 3/4, 4/25, respectively to groups of control, 20, 60 and 120 mg/kg bw/day) as a slight neuro-hormonal phenomenon in connection with the sexual function of inner genital organs (pro-oestrus).
In one female animal at 120 mg/kg bw/day (1/25), histological investigations revealed moderate hyperplasia of spleen consisting of erythroid, myeloid and lymphoid elements. The splenic hyperplasia affected the red pulp (erythroid, myeloid and megakaryocytes) and the white pulp (periarteriolar lymphoid sheath, number and size of lymphoid follicles and size of marginal zones). Based on the cytomorphology and the distribution of these cells and the preserved intact normal splenic structure, neoplastic diseases could be excluded.
One or both sided renal pyelectasia was detected in several case in each group as follows:
- Control: 7/26 male and 8/26 female
- 20 mg/kg bw/day: 7/7 male and 2/2 female
- 60 mg/kg bw/day: 5/5 male and 4/4 female
- 120 mg/kg bw/day: 9/26 male and 9/25 female
Pyelectasia without significant histological lesions (inflammation, degeneration, fibrosis etc.) is considered as a common finding in laboratory rats without toxicological significance.
Some individual histological findings were also seen in parental animals as follows:
- Focal fibrosis in the Glisson’s capsule (1/26 male at 120 mg/kg bw/day) could be in connection with the mechanical irritation of the affected lobe of liver (due to diaphragmatic hernia), without toxicological importance.
- Acute hemorrhage in the lungs (1/26 male at 120 mg/kg bw/day) and congestion in the thymus (1/26 male control, 1/1 male at 20 mg/kg bw/day) are considered to be in connection with the agonal circulatory disturbance and are individual disorder without toxicological relevance.
There was no morphological evidence of test item related acute or subacute injuries (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes or integumentary system.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A test item influence on the estrous cycle was not detected at any dose level (20, 60 or 120 mg/kg bw/day).
The estrous cycle was irregular in several parental female animals in each group – mainly in the control and high dose groups – during the two last weeks of an overall of 10 weeks pre-mating period.
Statistical significance was noted for the higher mean number of cycles, for the higher mean number of days in estrous and lower mean number of days in diestrus at 20 mg/kg bw/day when compared to the control.
The examined parameters of the estrous cycle were comparable in the control and 60 mg/kg bw/day groups.
At 120 mg/kg bw/day, the mean length of cycle was slightly longer than in the control group and also in the historical control data. However, there were no disturbances in reproductive performance (copulatory and fertility ability) of high dose females has not been influenced by this change in cycle length. Therefore, it was considered to be toxicologically not relevant.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm morphology, motility and sperm count were not adversely affected by the test item at 20, 60 or 120 mg/kg bw/day.
Statistical significances were detected at the slightly lower mean sperm count in each dose group and at the higher mean percentage of immotile sperm cells and sperms with separated head in parental male animals at 120 mg/kg bw/day when compared to the control.
Slight changes in the sperm count were judged to have little or no toxicological relevance as no effect was detected at the reproductive performance.
The percentage of motile sperms and sperm with normal morphology were comparable in all groups. Therefore, these statistically significant differences between the control and test item treated animals were indicative of biological variation and normal function of male genital organs at 120 mg/kg bw/day.

Reproductive performance:

no effects observed

Description (incidence and severity):

The reproduction performance of male and female animals (mating and fertility) was not selectively affected by the treatment with 20, 60 or 120 mg/kg bw/day with respect to their control.
In the male animals, a higher fertility index (percentage of male animals impregnating females) at 20 mg/kg bw/day and a lower copulatory index at 120 mg/kg bw/day were observed when compared to their control.
Similarly, in the female animals, the fertility index (percentage of pregnant female animals) was higher at 20 mg/kg bw/day and the gestation index (percentage of female giving birth) was lower at 120 mg/kg bw/day comparing to their control. The mean number of conceiving days was longer than in the control group at 120 mg/kg bw/day.
Test item effect was not presumed because data met well historical control value and in the lack of related findings in the reproductive parameters or reproductive organs. There were no biologically relevant differences between the control and test item treated animals in fertility indices.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

3

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs in male or female animals in F1 Cohort 1A and 1B generation in control, 20, 60 or 120 mg/kg bw/day groups. The behavior and physical condition of male and female animals were normal during the entire observation period.
Species specific signs were detected in some female animals of Cohort 1B as follows:
- porphyrin contaminated hairs around left eye: 1/20 at 20 mg/kg bw/day and 1/20 at 20 mg/kg bw/day between PND39-91 and PND 37-*109, respectively
- slight alopecia on the neck (both sides) 2/20 at 20 mg/kg bw/day between PND77-91
These signs are commonly observed in non-treated experimental rats and occurred only in the low dose group with low incidence. Therefore, they are considered to have no toxicological relevance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no mortality in F1 Cohort 1A and 1B animals in control, 20, 60 or 120 mg/kg bw/day groups (male or female) during the course of study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was depressed in F1 Cohort 1A animals at 60 mg/kg bw/day (male) and 120 mg/kg bw/day (male and female). In the presence of a dose-dependency this finding is considered treatment-related. The reduction in body weight compared to control was 6 % or less in the male animals at 60 mg/kg bw/day and was therefore considered to have little or no toxicological relevance.
The mean body weight and body weight gain were similar to their control in F1 Cohort 1A male animals at 20 mg/kg bw/day during the entire observation period.
At 60 mg/kg bw/day, the mean body weight of male animals remained below the control from PND49 up to termination on PND91 (minus 5-6 %) as the body weight gain was lower than in the control group from week 3 up to termination (on weeks 4, 6, 8 and 10). The summarized body weight gain of mid dose treated male animals was also lower than in the control group.
In the male animals at 120 mg/kg bw/day, the mean body weight was lower than in the control group from post-natal day 22 reaching statistical significance from post-natal day 29 up to and including PND91 (6-20 % reduction). The body weight gain of high dose treated male animals was also lowered with respect to the control at the end of first week and from post-natal day 32 up to and including PND 91 resulting in significantly lower mean summarized body weight gain (between PND22 and PND91).
In female animals at 20 and 60 mg/kg bw/day, the body weight development was comparable to their control although, the mean body weight gain was transiently significantly lowered at 60 mg/kg bw/day between post-natal days 22-25.
In the female animals at 120 mg/kg bw/day, the mean body weight was lower than in the control group on each measuring day from PND 22 up to PND 91. The mean body weight gain was below the control during the entire treatment period reaching statistical significance on weeks 1, 2, 5, 6, 7 and if summarized between PND22 and 91.

The body weight development was depressed in F1 Cohort 1B animals at 60 mg/kg bw/day and at 120 mg/kg bw/day (male and female). The reduction in body weight compared to control was 7 % or less in the female animals at 60 mg/kg bw/day and was therefore considered to have little or no toxicological relevance.
The mean body weight and body weight gain were predominantly similar to their control in F1 Cohort 1A male animals at 20 mg/kg bw/day during the entire observation period. A lowered mean body weight gain in this group was observed between postnatal days 63 and 70.
At 60 mg/kg bw/day, the mean body weight of male animals remained below the control from PND42 up to termination on PND105 (6-12 % reduction) as the body weight gain was lower than in the control group during the entire observation period reaching statistical significance between post-natal days 22-25, from PND36 to PND77 and between post-natal days 84-91, 98-105. The summarized body weight gain of mid dose treated male animals was also lower than in the control group.
In the male animals at 120 mg/kg bw/day, the mean body weight was lower than in the control group from post-natal day 25 up to and including PND105 (8-24 % reduction). The mean body weight gain of high dose treated male animals was lowered with respect to the control from PND22 up to and including PND 105, except from PND 25-29, resulting in significantly lower mean summarized body weight gain.
In the female animals at 20 mg/kg bw/day, the body weight development was comparable to their control from PND22 to PND105.
At 60 mg/kg bw/day, the mean body weight of female animals remained below the control on PND 63 and from PND77 up to PND105 (5-7 % reduction). Statistical significances with respect to the control was observed in the mean body weight gain between post-natal day 22-25 and between post-natal days 22-105.
The mean body weight was significantly lower than the control during the entire study period in female animals at 120 mg/kg bw/day (-6 to -14%). The reduction of mean body weight gain in this group reached statistical significance on PND25-29, PND32-36, PND42-49, PND56-63, and in the mean summarized body weight gain (PND22-105).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A reduced mean daily food consumption of F1 Cohort A male and female animals at 120 mg/kg bw/day was in accordance with the body weight changes during the entire observation period.
At 20 and 60 mg/kg bw/day, the mean daily food consumption of F1 Cohort 1A male and female animals was predominantly similar to their control. Statistical significance was detected at the lower mean daily food consumption at 60 mg/kg bw/day in male animals on weeks 8, 10 and in female animals on weeks 9, 10.
At 120 mg/kg bw/day, the mean daily food consumption was lowered in male animals from week 3 up to the end of the study.
In the female animals at 120 mg/kg bw/day, statistically significant difference with respect to the control was detected at the lower mean daily food consumption on weeks 1, 4, 5, 7 and 10.

The mean daily food consumption of F1 Cohort B male and female animals was reduced at 60 and 120 mg/kg bw/day during the observation period in full accordance with the body weight changes.
At 20 mg/kg bw/day, the mean daily food consumption of F1 Cohort 1B male and female animals was comparable to the control during the observation period (between PND22 and PND105).
Statistically significant difference with respect to the control was detected at the lower mean daily food consumption in male animals from week 3 up to week 6 and from week 8 up to termination (-8 to -11 %) and in female animals from week 5 up to week 11 (-11 to -15 %) at 60 mg/kg bw/day.
At 120 mg/kg bw/day, the mean daily food consumption was lower than in the control group from weaning up to termination (-10 to -20 %) except for treatment week 2 in male animals and from week 4 to week 11 in female animals (-11 to -15 %).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse changes in the examined hematological parameters in F1 Cohort 1A male or female animals at 20, 60 or 120 mg/kg bw/day.
In the male animals at 20 mg/kg bw/day, statistical significances were detected at the slightly higher mean hemoglobin concentration (HGB) when compared to the control.
At 60 mg/kg bw/day, higher mean hemoglobin concentration and hematocrit (HCT), higher mean corpuscular hemoglobin (MCH), elevated percentage of reticulocytes (RET) and longer mean activated thromboplastin time (APTT) were detected when compared to the control.
In the male animals at 120 mg/kg bw/day, the mean red blood cell count (RBC), mean hemoglobin concentration and hematocrit, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration (MCHC) were higher than in the control group.
In the female animals at 20 mg/kg bw/day, the mean percentage of eosinophil granulocytes (EOS) was lower and the mean hemoglobin concentration was higher than in the control group.
At 60 mg/kg bw/day, statistical significances with respect to the control were detected at the lower mean percentage of eosinophil granulocytes, at the higher mean hemoglobin concentration and mean corpuscular hemoglobin concentration as well as at the elevated mean percentage of reticulocytes in the female animals.
In the female animals at 120 mg/kg bw/day, the mean white blood cell count (WBC), the mean red blood cell count, hemoglobin concentration and hematocrit and mean corpuscular hemoglobin concentration were higher than in the control group.
Elevated level of some red blood cell (RBC, HGB, HCT) parameters may refer to a slight dehydration in male and female animals in accordance with lowered mean volume of urine only in female animals at 120 mg/kg bw/day.
Changes in WBC and RET were of small degree and not related to histopathological findings. Therefore, these changes were considered to be variations and toxicologically not relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 20, 60 or 120 mg/kg bw/day by (male or female).
The investigated clinical chemistry parameters were similar to their control in male animals at 20 mg/kg bw/day and in female animals at 20 and 60 mg/kg bw/day.
In male animals, statistically significant difference was detected with respect to the control at the lowered mean concentrations of creatinine (CREA) and glucose (GLUC) at 60 and 120 mg/kg bw/day and at the lower mean total protein (TPROT) at 120 mg/kg bw/day.
In female animals at 120 mg/kg bw/day, the mean creatinine concentration was lower than in the control. The other examined parameters were similar to their control.
The statistically significant differences in clinical chemistry parameters were considered to have little or no toxicological importance because values – mean and individual – corresponded well to the historical control values.

The thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the F1 Cohort 1A male or female animals at any dose levels.
There were no statistically significant differences with respect to their control in the FT4 and TSH concentrations in male or female animals at 20, 60 or 120 mg/kg bw/day levels. The FT3 concentrations were higher than in the control in male animals at 120 mg/kg bw/day and in female animals at 60 and 120 mg/kg bw/day. The differences from the control were with minor degree both in male and female animals and values met well the historical control range. There were no related changes in the thyroid glands (weights or histopathology investigations). Therefore, these changes were considered to be incidental ones and not related to treatment with the test item.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item related changes in the examined urine parameters in F1 Cohort 1A animals (male or female) at 20, 60 or 120 mg/kg bw/day.
Statistical significance with respect to the control was observed at the slightly lower mean volume of the urine in female animals at 60 and 120 mg/kg bw/day.
Since no related changes in other parameters in urine, clinical chemistry, or renal organ pathology were observed, changes in urine volume were considered to have little or no toxicological relevance. Additionally, similar findings were not observed in male animals. The examined urine parameters were comparable in male animals in the control and 20, 60 and 120 mg/kg bw/day groups.

Sexual maturation:

no effects observed

Description (incidence and severity):

The examined parameters of sexual maturity were similar in the control and 20, 60 and 120 mg/kg bw/day in F1 Cohort 1A and 1B male and female animals.
The balano-preputional separation was completed in all F1 Cohort 1A and 1B male animals – control, 20, 60 and 120 mg/kg bw/day – on post-natal day 25, although, the mean body weight was slightly but significantly lower with respect to the control in male animals at 120 mg/kg bw/day on PND25. This minor difference from control (-6 %) was considered to be toxicologically not relevant.
In the F1 Cohort 1A and 1B female animals, post-natal days of vaginal patency was slightly later than the control at 60 and 120 mg/kg bw/day. The post-natal day of the appearance of the first cornified vaginal smear and the interval between days of vaginal patency and first cornified smear were similar in all groups (control, 20, 60 and 120 mg/kg bw/day).

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no differences from the control in the body weight on PND4, and in absolute and normalized anogenital distance in male offspring.
In the female offspring, statistical significances with respect to the control were detected at lower mean body weight on PND4 (120 mg/kg bw/day), and at shorter mean absolute anogenital distance (20 and 60 mg/kg bw/day), and at the lower mean normalized anogenital distance (60 mg/kg bw/day). The changes in body weight and anogenital distances were of minor degree and values met well the historical control range. Therefore, these changes in examined parameters of offspring’s development were considered to be of little or no toxicological relevance.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipples/areoles were not visible in any of the examined male offspring in the control, 20, 60 or 120 mg/kg bw/day groups on post-natal day 13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring
There was no test item related adverse effect in the weights of examined organs (absolute and relative to body and brain weights) in male and female F1 offspring at the necropsy at weaning.
Statistical significance with respect to the control were detected at the lower mean brain weight (absolute) in male offspring at 120 mg/kg bw/day. Although not statistically significant, the absolute mean weights of brain, spleen and thymus and the weights relative to body and brain weight of spleen and thymus are below the control value in male offspring at 20 and 60 mg/kg bw/day. However, in the absence of a dose dependency and the low degree of deviation compared to control this finding is considered incidental and not related to treatment.
In the female offspring at 20 mg/kg bw/day, the weights of spleen (absolute and relative to body and brain weights) were slightly but statistically significantly lower than in the control.
At 60 mg/kg bw/day, the weights of examined organs (absolute and relative to body and brain weights) were comparable with their control in female offspring.
The absolute mean weights of brain, spleen and thymus remained below the control value in female offspring at 120 mg/kg bw/day. Similarly, the spleen weights relative to body and brain weights and thymus weight relative to brain weight were statistically significantly lowered comparing to their control at 120 mg/kg bw/day.
Statistical significances noted at the absolute or relative weights of brain, spleen or thymus in female animals were probably indicative of biological variation at 20 or 120 mg/kg bw/day in the lack of dose relevance. At 120 mg/kg bw/day, the changes in organ weights were partially due to the lowered mean fasted body weight (statistically not significant) of female animals.
Therefore, test item effect in the changes in organ weights cannot be proved with the results of this study.

F1 Cohort 1A
The weights of the examined organs were not adversely affected in F1 Cohort 1A male and female animals at 20, 60 or 120 mg/kg bw/day.
The fasted mean body weight was dose-dependently lowered in male animals at 60 and 120 mg/kg bw/day comparing to the control. Therefore, the absolute weights of several organs remained below the control while the weight of these organs relative to body weight exceeded the control or there was no significant difference from the control. There were no accompanying changes in clinical pathology parameters or histological lesions in these organs. Therefore, weight changes of these organs were considered to be of little or no toxicological relevance.
At 20 mg/kg bw/day, statistically significant difference with respect to the control was observed at the lower mean weight of thymus and thyroid glands (absolute and relative to brain weights, both organs) and at the higher mean weight of submandibular lymph nodes (absolute and relative to body and brain weights) in male animals.
In the male animals at 60 mg/kg bw/day, the fasted mean body weight and thymus weight (absolute and relative to brain weight, both organs) and the mean thyroid weights (absolute and relative to body and brain weights) were lower, and the mean weight of submandibular lymph nodes relative to body weight was higher than in the control group. The organ weight relative to body weight exceeded the control in case of brain, kidneys, heart, spleen, testes, epididymides.
In male animals at 120 mg/kg bw/day, significantly lowered mean fasted body weight, lowered mean weights of brain, liver, kidneys, heart, thymus, spleen, epididymides, seminal vesicles, prostate and thyroid glands were detected when compared to their control. The mean weights relative to body weight of brain, kidneys, heart, testes, epididymides, adrenal glands, and submandibular lymph nodes were higher than in the control group. Weights relative to brain weight were significantly lowered at the mean body weight, liver, thymus, seminal vesicles, prostate and thyroid glands.
In the female animals at 20 mg/kg bw/day group, the weights of all examined organs were comparable with their control (absolute and relative to body and brain weights).
At 60 mg/kg bw/day, statistically significant difference with respect to the control was observed at the lower mean weight of thymus (absolute) and at the higher mean weight of spleen relative to body weight in female animals.
At 120 mg/kg bw/day, the mean fasted body weight, and the mean weights of brain, thymus, adrenal and thyroid glands were below the control value in female animals. Statistical significance with respect to the control was observed at the higher mean weights of liver, kidneys, heart and spleen, each relative to body weight. The thymus weight relative to brain weight was lower than in the control group in high dose treated female animals.
Morphological changes were not detected during the histopathological examination. Hematology investigations as well as clinical chemistry parameters did not reveal test item related abnormalities. Therefore, changes in organ weights were judged to have little or no toxicological relevance.

F1 Cohort 1B
The weights of the examined organs were not adversely affected in F1 Cohort 1B male and female animals at 20, 60 or 120 mg/kg bw/day. The fasted mean body weight was dose-dependently lowered in male and female animals at 60 and 120 mg/kg bw/day comparing to the control. Therefore, the absolute weights of several organs remained below the control while the weight of these organs relative to body weight exceeded the control or there was no significant difference from the control.
At 20 mg/kg bw/day, statistically significant difference with respect to the control was observed at the lower mean weights of seminal vesicles and prostate (absolute and relative to brain weight) in male animals.
In male animals at 60 mg/kg bw/day, significantly lowered mean fasted body weight (absolute and relative to brain weight) and lowered mean weights of brain (absolute), seminal vesicles, (absolute and relative to brain weight), prostate (absolute and relative to brain weight) and pituitary (absolute) were observed when compared to the control. The weights of brain, testes and epididymides relative to body weight exceeded the control.
At 120 mg/kg bw/day, the mean fasted body weight and mean absolute weight of all examined organs (brain, testes, epididymides, seminal vesicles, prostate and pituitary) were below the control value. Statistical significances with respect to the control were also noted for the higher mean weight of brain and testes relative to body weight as well as for the lower mean fasted body weight, weights of epididymides, seminal vesicles and prostate relative to brain weight.
In the female animals at 20 mg/kg bw/day, the weight of examined organs were comparable with their control.
At 60 mg/kg bw/day, statistically significant difference with respect to the control was observed at the lower mean the mean fasted body weight, brain weight, thymus weight and pituitary weight in female animals. The relative organ weights (to body and brain weights) were comparable to control.
In the female animals at 120 mg/kg bw/day, statistically significant difference with respect to the control was observed at the lower mean fasted body weight, at the lower mean absolute weight of brain, thymus, uterus and pituitary. The brain weight and spleen weight relative to body weight exceeded the control while the body weight and uterus weight both relative to brain weight were below the control in high dose administered female animals.
Although the above-mentioned differences with respect to the control achieved statistical significances in the weights of examined organs, these were considered to be of little or no toxicologically significance in the absence of related morphological (histopathology) findings and corresponding findings in F1 Cohort 1A.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring
There were no test-item-related macroscopic alterations in F1 offspring subjected to gross pathological examination before weaning or at weaning.
Some common sporadic necropsy findings were detected in pups subjected to necropsy before weaning:
- smaller than normal: 2/50 at 20 mg/kg bw/day, 1/23 at 60 mg/kg bw/day
- pale: 2/50 at 20 mg/kg bw/day,
- empty stomach, autolyzed visceral organs: 1/16 at 120 mg/kg bw/day
- cannibalized: 2/16 at 120 mg/kg bw/day
There were no macroscopic findings in any other ones.
At weaning, there were no macroscopic findings in any of the examined pups: 109, 133, 102 and 55 pups in groups of control, 20, 60 and 120 mg/kg bw/day, respectively.

F1 Cohort 1A
Macroscopic alterations related to the effect of the test item were not detected in F1 Cohort 1A male or female animals at 20, 60 or 120 mg/kg bw/day at the necropsy.
Gross necropsy observations revealed common findings occurring also in non-treated experimental rats as follows:
Male animals:
- control: right or both sided renal pyelectasia: 13/20
- 20 mg/kg bw/day: right or both sided renal pyelectasia: 12/20, mustard seed size hole on the surface of right-side kidney 1/20, brownish red colored thymus: 1/20, thymic hemorrhages: 1/20
- 60 mg/kg bw/day: right or both sided renal pyelectasia: 8/20, pale liver: 1/20
- 120 mg/kg bw/day: right or both sided renal pyelectasia: 12/20,
Female animals:
- control: right or both sided renal pyelectasia: 8/20, slight or moderate hydrometra: 7/20, reddish formation at bifurcation of uterus: 1/20
- 20 mg/kg bw/day: one or both sided renal pyelectasia: 6/20, slight, moderate or marked hydrometra: 9/20
- 60 mg/kg bw/day: right or both sided renal pyelectasia: 6/20, moderate or marked hydrometra: 8/20, pin-head size, thymic hemorrhages, right side: 1/20, adrenal gland: brownish gray formation, 3x5 mm, right side: 1/20
- 120 mg/kg bw/day: right or both sided renal pyelectasia: 6/20, dilated vesicle like and liquid filled kidney (1/20), dilated ureter (1/20), slight, moderate or marked hydrometra: 7/20
Pyelectasia and hydrometra are common observations in untreated experimental rats of this strain with similar age and similar occurrence noted in this study. Hydrometra (i.e., dilatation of uterine horns with clear liquid content) is related to the female sexual cycle and is a physiological phenomenon. In the lack of related inflammatory or other pathological signs, these findings were judged to be toxicologically not relevant and not test item related as no dose response was noted.
Discolored thymus and thymic hemorrhages are also frequently observed findings in experimental rat in connection with the exsanguination procedure.
Necropsy findings in single animals (kidneys, ureter, liver, uterus) were individual alterations occurring commonly in this strain of experimental rats.

F1 Cohort 1B
Macroscopic alterations related to the treatment with the test item were not detected in F1 Cohort 1B male or female animals at 20, 60 or 120 mg/kg bw/day at the necropsy.
In male animals, necropsy observations revealed right or both sided renal pyelectasia (7/20, 13/20, 11/20 and 9/20 respectively to groups of control, 20, 60 and 120 mg/kg bw/day).
In one male animal at 120 mg/kg bw/day (1/20) dilated and enlarged kidney was observed at the necropsy observations.
In female animals, necropsy observations revealed the following findings:
- control group: right or both sided pyelectasia in kidneys (4/20), Hernia diaphragmatica (1/20), slight, moderate or marked hydrometra (10/20)
- 20 mg/kg bw/day: right or both sided pyelectasia in the kidneys (6/20), slight, moderate or marked hydrometra (5/20), alopecia on the skin on the neck right side or both sides (2/20)
- 60 mg/kg bw/day: right or both sided pyelectasia in the kidneys (7/20); slight, moderate or marked hydrometra (6/20)
- 120 mg/kg bw/day: right or both sided pyelectasia (5/20), marked or moderate hydrometra (5/20) and formation in the left side uterine horn
Pyelectasia, hydrometra and alopecia on the skin are common findings in experimental rats of this strain and age. Hydrometra (i.e., dilatation of uterine horns with liquid content) related to the female sexual cycle, is a physiological phenomenon. In the lack of related inflammatory or other pathological signs, these findings were judged to be toxicologically not relevant and not test item related as no dose response was noted.
Formulation in the uterine horn was considered to be individual macroscopic alterations and not related to treatment.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Histological examinations did not reveal pathologic alterations in the organs or tissues of F1 Cohort 1A male or female animals at 120 mg/kg bw/day.
In the F1 Cohort 1A male animals in the control and 120 mg/kg bw/day groups the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in all cases.

The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and high dose treated animals.
The histological picture of epididymides, prostate, seminal vesicles, and coagulating glands was normal in all cases as well.
In the F1 Cohort 1A female animals of the control and 120 mg/kg bw/day groups, the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases.
Quantitative examinations of ovaries did not reveal test item related changes in the number of developing follicles, corpora lutea or in the number of follicular atresia at the examined level of section of F1 Cohort 1A female animals at 120 mg/kg bw/day.
The mean number of primordial and primary follicles as well as corpora lutea was similar in F1 Cohort 1A female animals in the control and at 120 mg/kg bw/day.
The mean number of secondary and tertiary follicles was significantly higher and the mean number of follicular atresia was lower in F1 Cohort 1A female animals at 120 mg/kg bw/day compared to the control. However, the values were within the historical control range.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
Dilatation of uterine horns was observed in control (5/20) and high dose (7/20) groups. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (proestrus phase) of uterus without pathological significance.
One or both sided pyelectasia was seen in several F1 Cohort 1A male and female animals:
- control: 13/20 male and 8/20 female
- 20 mg/kg bw/day: 11/12 male and 6/6 female
- 60 mg/kg bw/day: 8/8 male and 6/6 female
- 120 mg/kg bw/day: 12/20 male and 6/20 female
Pyelectasia without other histopathological lesions (for example degeneration, inflammation, fibrosis etc.) is considered as an individual disorder without toxicological significance.
In one male animal at 20 mg/kg bw/day (1/12), cortical cyst was detected. This finding is considered incidental as it occurred only in a single animal at the low dose.
In one female animal in 120 mg/kg bw/day group (1/20), one side hydronephrosis was observed in accordance with necropsy findings (dilated, vesicle like and liquid filled kidney, dilated ureter). This lesion occurs also in non-treated experimental rats and has no toxicological relevance in this study.
Acute hemorrhage in the thymus (2/2 male at 20 mg/kg bw/day, 1/1 female at 60 mg/kg bw/day) occurred with low incidence and is considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Focal necrosis in one side adrenal gland in one female animal at 60 mg/kg bw/day (1/1) could be considered as individual lesion without toxicological significance.
There was no morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes, integumentary system.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous cycle
The estrous cycle was not adversely affected in the F1 Cohort 1A female animals at 20, 60 or 120 mg/kg bw/day groups. The examined parameters of the estrous cycle were comparable in the control and 20 and 60 mg/kg bw/day.
Statistical significances were observed at longer mean length of cycle, lower mean number of days in pre-estrous and at higher mean number of days in diestrous in female animals 120 mg/kg bw/day group when compared to control. These differences with respect to the control were with minor degree for this highly variable biological system and were therefore considered to be not treatment-related or adverse.
The estrous cycle was not adversely affected in the F1 Cohort 1B female animals at 20, 60 or 120 mg/kg bw/day groups. The examined parameters of the estrous cycle were comparable in the control and 20, 60 and 120 mg/kg bw/day.

Sperm parameters
Sperm examinations did not show any test item related or adverse influence on the sperm cells at 20, 60 or 120 mg/kg bw/day in Cohort 1A male animals.
Statistical significance was detected at the slightly higher mean percentage of immotile sperm cells in Cohort 1A male animals at 60 and 120 mg/kg bw/day when compared to the control.
The sperm count, percentage of motile sperms and sperm with normal morphology were comparable in all groups (control, at 20, 60 or 120 mg/kg bw/day). Therefore, the statistically significant difference between the control and test item treated animals were judged to be indicative of biological variation and normal function of male genital organs at 60 and 120 mg/kg bw/day.

Splenic Lymphocyte Subpopulation Analysis
There were no adverse changes in the splenic lymphocyte subpopulation in male or female animals in F1 Cohort 1A animals in 20, 60 or 120 mg/kg bw/day groups.
A test item-related effect on the dose-dependent changes in percentages of B and T cells at 20, 60 and 120 mg/kg bw/day cannot be excluded. However, almost all mean and individual values were within the historical control ranges and statistical significances were mainly due to the relatively low or high values respectively in the control group.
Statistically significant difference with respect to the control was detected at the higher mean percentage of B cells and at the lower mean percentage of T cells in male animals at 20, 60 and 120 mg/kg bw/day. The mean percentage of cytotoxic T cells was lower than in the control group in male animals at 120 mg/kg bw/day.
At 120 mg/kg bw/day, the mean percentage of B cells was higher and the mean percentage of cytotoxic T cells was lower than in the control group in the female animals
These minor changes were considered to be indicative of biological variation as values mostly met well the normal ranges even at 120 mg/kg bw/day.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, administration of Potassium Cyanate via oral gavage did not adversely influence the reproductive performance (gonad function, estrous cycle, mating behavior, conception, parturition, gestation and lactation) in parental male and female Han:WIST rats at 20, 60 and 120 mg/kg bw/day.
Doses of 120 and 60 mg/kg bw/day caused reduction of body weight and food consumption in male or female animals in P generation.
At 20 mg/kg bw/day of P generation, there were no signs of systemic toxicity (male or female animals).
The offspring’s development was not adversely affected at 20, 60 or 120 mg/kg bw/day from birth to post-natal day 21.
F1 adult (F1 Cohort 1A, Cohort 1B)
Reduced body weight development and food consumption were detected at 120 mg/kg bw/day in F1 adult animals. Growth, development, sexual maturation and thyroid hormone levels of the F1 offspring (Cohort 1A and 1B) to adulthood following administration via oral gavage were undisturbed at 20, 60 and 120 mg/kg bw/day. No adverse effects on clinical, macroscopic or microscopic pathological parameters were recorded in all dose groups of Cohort 1A and 1B.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity (parental male): 60 mg/kg bw/day
NOAEL for systemic toxicity (parental female): 120 mg/kg bw/day
NOAEL for reproductive performance (male and female): 120 mg/kg bw/day
NOAEL for F1 Offspring: 120 mg/kg bw/day
NOAEL for F1 Adults: 60 mg/kg bw/day

Executive summary:

The subject of this study was investigating the reproductive toxicity of Potassium Cyanate by conducting the Extended One-Generation Reproduction Toxicity Study in the rat according to OECD 443 as requested by the European Chemical Agency (ECHA). The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.

The aim of the One-generation Reproduction Toxicity Study was to provide an evaluation of the pre- and post-natal effects of the test item Potassium Cyanate on development as well as a thorough evaluation of systemic toxicity in male and female animals, in pregnant and lactating females and in young and adult offspring when repeatedly administered orally by gavage to animals at doses of 20, 60 and 120 mg/kg bw/day compared to control animals.

The effect of the test item was examined on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation, lactation and weaning (P generation) and on the offspring viability, neonatal health and mortality, growth and development of the offspring (F1 generation; Cohort 1A and Cohort 1B) to adulthood following oral administration by gavage. Four groups of Han:WIST rats (n= 26/sex/group) were administered with the test item orally by gavage once a day at 0 (vehicle), 20, 60 and 120 mg/kg bw/day doses corresponding to concentrations of 0, 4, 12, and 24 mg/mL in parental generation. The application volume was 5 mL/kg bw. Control animals received the vehicle, distilled water, only.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. Analytical control of dosing solutions was performed five times during the study. Potassium Cyanate concentration varied between 98.3 % and 103 % of nominal concentrations, thereby confirming the proper dosing of animals.

All animals of the parent (P) generation were dosed prior to mating (10 weeks) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 127-129 days). Dams were exposed through the mating and gestation periods and at least up to lactation day 21 (altogether for 114-135 days). Non-pregnant and not delivered females were administered for 105 or 114 days and moribund animal for 43 days.

Clinical observations (clinical signs, body weight, food consumption, estrous cycle) and pathology (clinical pathology and organ pathology) examinations were performed on parental (P) animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems. Estrous cycle was monitored by examining vaginal smears before the mating for two weeks and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 post-partum. All F1 offspring were observed individually for the health, growth, development and function up to and including post-natal day 21 (clinical signs, body weight, surface righting reflex, pinna detachment, eye opening, anogenital distance, nipple retention).

Twenty F1 animals/sex/group were randomly selected for Cohort 1A in each group and twenty F1 animals/sex/group were selected for Cohort 1B in the control, low, mid and high groups on post-natal day 21 for follow-up examinations. Dosing of F1 offspring selected for follow-up examinations (Cohort 1A and Cohort 1B) begun on post-natal day 22 and treatment was continued up to the day before the necropsy, i.e., up to PND 91-113 and PND98-114. F1 offspring (Cohort 1A and Cohort 1B) were administered and observed identically to parental animals – clinical signs, body weight, food consumption, estrous cycle, clinical pathology and organ pathology. Sexual maturity of offspring was investigated by observation of balano-preputional separation, vaginal patency (Cohort 1A and Cohort 1B) and appearance of first cornified vaginal smear (Cohort 1A).

Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 10 parent (P) animals/sex/ group at termination, in surplus pups at PND 4 (pooled by litters), from F1 pups not selected for cohorts on PND 22 and from 10 adult F1 Cohort 1A male and female animals/group at termination. Thyroid hormone levels were determined in adult animals (P, F1 Cohort 1A) and in PND22 F1 offspring. All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. The F1 pups not selected for Cohorts were also subjected to gross necropsy on PND 22 or shortly thereafter. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where it was feasible. Special attention was paid to the organs and tissues of the reproductive system for P or F1 animals. Selected organ weights were determined in adult animals (P, F1) and in F1 offspring not selected for Cohorts on PND22 or shortly thereafter. Sperm parameters were determined in all adult male animals in control and high dose groups in P generation and in F1 generation (Cohort 1A). Splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female animals Cohort 1A animals from each group.  Full histopathology examinations were performed on the organs and tissues of adult animals (P, F1 Cohort 1A) in control and high dose groups with special emphasis on sexual organs and tissues. Reproductive organs were also processed and examined histologically in non-mated and non-pregnant female animals and their mating partners in the mid dose group. In addition, organs showing macroscopic findings were also processed and examined histologically in adult animals in low or mid dose groups (P, F1 Cohort 1A). A quantitative evaluation of primordial, small growing (secondary and tertiary) follicles, as well as corpora lutea was performed in F1 Cohort 1A female animals in the control and 120 mg/kg bw/day groups.

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (distilled water) only.

Results

Analytical control of dosing formulations

The concentration of the test item in the dosing formulations used for animal’s treatment was checked five times during the study. Concentration of Potassium Cyanate in the dosing formulations varied in the acceptable range between 98 % and 103 % of the nominal values and formulations were homogenous thereby confirming the proper dosing of parental animals (P) and F1 generations.

Parental (P) generation

Mortality

There was no mortality in male and female animals in control, 20, 60 or 120 mg/kg bw/day groups.

One female animal in the control group of the parental generation (1/26) was early euthanized in moribund state on Day 43 due to an individual disease.

Clinical observation

Clinical signs related to the test item were not detected at 20, 60 or 120 mg/kg bw/day at the daily or detailed weekly clinical observations.

Body weight and body weight gain

The body weight development was depressed in parental male animals at 60 and 120 mg/kg bw/day from Days 14 and 7, respectively, up to the end of the study and in female animals administered with 120 mg/kg bw/day from Day 28 and during the gestation and lactation period.

Slight changes in the mean body weight of male animals at 60 mg/kg bw/day and in female animals at 120 mg/kg bw/day during the gestation and lactation periods were judged to be toxicologically irrelevant as the difference with respect to the control remained less than 10 %.

Food consumption

The food consumption was reduced in parental male animals at 60 and 120 mg/kg bw/day and in female animals at 120 mg/kg bw/day consistently during the entire treatment period. The reduction was dose related in male animals and was in full accordance with body weight development in both sexes.

Estrous cycle

A test item influence on the estrous cycle was not detected at any dose level (20, 60 or 120 mg/kg bw/day).

Delivery data of dams

The examined parameters of pregnancy and delivery were not adversely influenced by Potassium Cyanate at 20, 60 or 120 mg/kg bw/day.

Reproductive performance

The reproduction performance of male and female animals (mating   and fertility) was not selectively affected by the treatment with 20, 60 or 120 mg/kg bw/day with respect to their control.

Urinalysis

The examined urine parameters were not adversely affected in male and female animals at 20, 60 or 120 mg/kg bw/day.

Clinical pathology

There were no test item related adverse changes in the examined urine, hematological, blood coagulation or clinical chemistry parameters in parental male or female animals at 20, 60 or 120 mg/kg bw/day.

Thyroid hormones

The serum thyroid hormone (FT3, FT4 and TSH) levels were not affected by the test item in parental male and female animals (20, 60 and 120 mg/kg bw/day) and in offspring sampled on post-natal day 22.

Necropsy

Necropsy observations did not reveal alterations related to the test item treatment in the organs or tissues of male or female animals at 20, 60 or 120 mg/kg bw/day.

Organ weight

There were no adverse test item related alterations in the weights of the examined organs in male or female animals at 20, 60 or 120 mg/kg bw/day.

Sperm examinations

Sperm morphology, motility and sperm count were not adversely affected by the test item at 20, 60 or 120 mg/kg bw/day.

Histopathology

The investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviduct, uterus cervix, vagina) were histologically normal and characteristic of the sexually mature organism in all parental (P) animals at 120 mg/kg bw/day.

Potassium Cyanate did not cause any toxic or other test item related lesions detectable by histological examinations of the investigated organs of experimental male and female animals.

Offspring

The offspring’s development was not adversely affected at 20, 60 or 120 mg/kg bw/day from birth to post-natal day 21. No effect on the mortality, clinical signs, body weight, development (surface righting reflex, pinna detachment or eye opening), anogenital distance, nipple retention (male only), necropsy findings or organ weight were detected.

F1 generation (Cohort 1A and Cohort 1B)

Mortality

There was no mortality in F1 Cohort 1A or Cohort1B animals in the control, 20, 60 or 120 mg/kg bw/day groups (male or female) during the course of study.

Clinical observation

The behavior and physical condition of F1 Cohort 1A and Cohort 1B animals was normal (characteristic of the species and strain) at each dose level (0, 20, 60 or 120 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.

Body weight and body weight gain

The body weight development was depressed in F1 Cohort 1A and Cohort 1B animals at 60 mg/kg bw/day or 120 mg/kg bw/day.

The reduction in body weight compared to control was 6 % or less in the Cohort 1A male and Cohort 1B female animals at 60 mg/kg bw/day and was therefore considered to have little or no toxicological relevance.

Food consumption

A reduced mean daily food consumption of F1 Cohort A male and female animals at 120 mg/kg bw/day and in F1 Cohort B male and female animals at 60 and 120 mg/kg was in accordance with the body weight changes during the entire observation period (7 % or less reduction).

Sexual maturity

The examined parameters of sexual maturity (balano-preputial separation in males and vaginal patency in females) were similar in the control and 20, 60 and 120 mg/kg bw/day in F1 Cohort 1A and Cohort 1B male and female animals

Estrous cycle

The estrous cycle was not adversely affected in the F1 Cohort 1A or Cohort 1B female animals at 20, 60 or 120 mg/kg bw/day groups.

Clinical pathology

Pathologic alterations were not detected at the evaluation of clinical pathology (urine, hematology and blood coagulation, clinical chemistry) parameters in F1 Cohort 1A male or female animals at 20, 60 or 120 mg/kg bw/day.

Thyroid hormones

The thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the F1 Cohort 1A male or female animals at any dose level.

Necropsy

Macroscopic alterations in accordance with the effect of the test item were not detected in F1 Cohort 1A or in F1 Cohort 1B (male or female) animals at 20, 60 or 120 mg/kg bw/day at the necropsy.

Organ weight

The weights of the examined organs were not adversely affected in F1 Cohort 1A or F1 Cohort 1B (male and female) animals at 20, 60 or 120 mg/kg bw/day.

Sperm examinations

Sperm examinations did not reveal any test item related effect on the sperm cell morphology and motility and total sperm count at 20, 60 and 120 mg/kg bw/day in F1 Cohort 1A male animals.

Histopathology

Histological examinations did not reveal pathological alterations in the organs or tissues of F1 Cohort 1A male or female animals at 120 mg/kg bw/day.

Quantitative examinations of ovaries did not reveal test item related changes in the number of developing follicles, corpora lutea or in the number of follicular atresia at the examined level of section of F1 Cohort 1A female animals at 120 mg/kg bw/day.

Splenic Lymphocyte Subpopulation Analysis

There were no adverse test item related changes in the splenic lymphocyte subpopulation in male or female animals in F1 Cohort 1A animals at 20, 60 or 120 mg/kg bw/day.

 

Under the conditions of the present study, administration of Potassium Cyanate via oral gavage did not adversely influence the reproductive performance (gonad function, estrous cycle, mating behavior, conception, parturition, gestation and lactation) in parental male and female Han:WIST rats at 20, 60 and 120 mg/kg bw/day. Doses of 120 and 60 mg/kg bw/day caused reduction of body weight and food consumption in male or female animals in P generation. At 20 mg/kg bw/day of P generation, there were no signs of systemic toxicity (male or female animals). The offspring’s development was not adversely affected at 20, 60 or 120 mg/kg bw/day from birth to post-natal day 21.

F1 adult (F1 Cohort 1A, Cohort 1B)

Reduced body weight development and food consumption were detected at 120 mg/kg bw/day in F1 adult animals. Growth, development, sexual maturation and thyroid hormone levels of the F1 offspring (Cohort 1A and 1B) to adulthood following administration via oral gavage were undisturbed at 20, 60 and 120 mg/kg bw/day. No adverse effects on clinical, macroscopic or microscopic pathological parameters were recorded in all dose groups of Cohort 1A and 1B.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity (parental male): 60 mg/kg bw/day

NOAEL for systemic toxicity (parental female): 120 mg/kg bw/day

NOAEL for reproductive performance (male and female): 120 mg/kg bw/day

NOAEL for F1 Offspring: 120 mg/kg bw/day

NOAEL for F1 Adults: 60 mg/kg bw/day