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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 Jan 2016 to 09 Sept 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
03 November 2015
Limit test:
no

Test material

Constituent 1
Test material form:
solid: crystalline
Details on test material:
Identification: Nocolok Cs Flux (SM)
Chemical name (IUPAC), synonym or trade name: Cesium potassium fluoroaluminate
CAS Number: 566947-29-3
Molecular formula: K(x)Cs(y)AlF(z)
Appearance: White powder
Batch: MPA 22002a
Purity/Composition: >99%
Test item storage: At room temperature
Specific details on test material used for the study:
- Stable under storage conditions until 31 July 2016 (expiry date)

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. WIL Research Europe B.V. has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of
reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Age at start pretest Females: approximately 10-12 weeks.
Age at start F0-treatment Males: approximately 10-12 weeks; Females: approximately 12-14 weeks.

- Housing:
- Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
- Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
- Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
- Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage.
Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Lactation Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.

- Diet: ad libitum
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: 25 Jan 2016 To: 26 April 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item.

VEHICLE: water
- Rationale: Based on trial formulations performed at facilities
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (04 March 2016), according to a validated method (Test Facility Study No.511280). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were exposed for 50-54 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were exposed for 42 days.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were based on results of a 28-day dose range finding study.
Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 10 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Examinations

Parental animals: Observations and examinations:
Mortality / Viability At least twice daily (early in the morning and close to the end of the working day)

CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations (clinical signs and arena) were conducted and functional observations were started at least 1 hour (± 30 min) after dosing.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4).
For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure (prior to first exposure) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

FUNCTIONAL OBSERVATIONS: Yes
The following tests were performed on the selected 5 animals/sex/group:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).

BLOOD: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

HAEMATOLOGY: The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant, using the ADVIA® 2120i Hematology System (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
White blood cells (WBC), Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets

CHEMICAL BIOCHEMISTRY: Yes

Blood Sampling for Thyroid Hormone Analysis
F0-generation, males and females:
End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of treatment (including all males that failed to sire).
Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retroorbital sinus and collected into serum tubes (Greiner Bio-One GmbH, Kremsmünster, Austria).
After clotting and centrifugation, serum was used as listed below.
Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum for measurement of thyroid-stimulating hormone (TSH).
Females: The serum was stored for possible measurement of thyroxine (T4) and/or thyroidstimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Litter observations:
Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/2000/7).

- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.

- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND 1 and 14. Sex ratio
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

- Blood Sampling for Thyroid Hormone Analysis
F1-generation, PND 4 pups: From 2 surplus pups per litter at culling (if possible). Blood samples from the 2 pups per litter were collected into one serum tube.
Blood samples were collected by decapitation, between 7.00 and 10.30 a.m.. Blood samples from the 2 pups per litter (0.4 mL in total) were collected into one serum tube (Greiner Bio-One GmbH, Kremsmünster, Austria) for possible future measurement of thyroxine (T4).
After clotting and centrifugation, serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months were discarded.

F1-generation, PND 13-15 pups:
From 2 pups per litter (if possible from one male and one female) at planned necropsy.
As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m.. Blood was collected into serum tubes.
After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150 μL serum for measurement of thyroxine (T4) and the remaining volume of serum for measurement of thyroid-stimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.
Postmortem examinations (parental animals):
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Necropsy was conducted on the following days:
- Males Following completion of the mating period (a minimum of 28 days of dose administration).
- Females which delivered PND 14-16.
- Females which failed to deliver (nos. 44 and 80): Post-coitum Days 42 (females with evidence of mating)
- Females with total litter loss (nos. 76): Within 24 hours of litter loss.

MACROSCOPIC EXAMINATION
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The number of implantation sites were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution.
Identification marks: not processed (Nasopharynx), Adrenal glands (Esophagus), (Aorta), Ovaries, Brain - cerebellum, mid-brain, cortex (7-levels), (Pancreas), Caecum Peyer's patches [jejunum, ileum] if detectable Cervix Pituitary gland, Clitoral gland, Preputial gland, Colon, Prostate gland,
Coagulation gland, Rectum, (Cowper’s gland), (Salivary glands - mandibular, sublingual),Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, Eyes (with optic nerve (if detectable) and Harderian gland), Skeletal muscle, (Skin), Mammary gland area (males and females), Spinal cord -cervical, midthoracic, lumbar
Femur including joint Spleen, (Glans penis), Sternum with bone marrow, (Levator ani plus bulbocavernosus muscle, complex (LABC)), Stomach, Testes, Heart, Thymus, Ileum ,Thyroid including parathyroid if detectable, Jejunum, (Tongue), Kidneys, Trachea, (Lacrimal gland, exorbital), Urinary bladder, (Larynx), Uterus, Liver, Vagina, Lung, infused with formalin, All gross lesions, Lymph nodes - mandibular, mesenteric
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHT:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
For Selected 5 animals/sex/group:
Adrenal glands, Brain, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix).
For all remaining animals:
Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavernosus muscle complex (LABC), Testes, Thyroid.
Absolute organ weights and organ to body weight ratios were reported.

HISTOPATHOLOGY:
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- Stomach and Adrenal glands of all selected 5 animals of Groups 2 and 3 (males and females), based on treatment-related changes in these organs in Group 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile (see table below) or which died before mating to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
- All gross lesions of all animals (all dose groups).
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
A peer review on the histopathology data was performed by a second pathologist.
Postmortem examinations (offspring):
Pups, younger than 7 days were killed by decapitation.
On PND 4 (at culling), from 2 pups per litter blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m.. Blood samples from the 2 pups per litter werecollected into one serum tube.
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination. Blood was collected into serum tubes.
All remaining pups (PND 7-15) were sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater, The Netherlands) by intraperitoneal (ip) injection.
Pups found dead during the weekend were fixed in an identified container containing 70% ethanol (Klinipath, Duiven, The Netherlands) because they were not necropsied on the sameday.
Necropsy was conducted on the following days:
Condition Day of necropsy:
Culling: PND 4.
Terminal sacrifice: PND 13-15.
Spontaneous deaths: As soon as possible after death and always within 24 hours.

MACROSCOPIC EXAMINATION
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling was preserved in 10% buffered formalin.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence no milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Offspring viability indices:
Viability index (%) = [(Number of live offspring on Day 4 before culling)/(Number live offspring on Day 1 after littering)]x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
The daily clinical observations showed no treatment-related findings.
The weekly observations outside the home cage in a standard arena did not show any additional clinical signs of behavioural changes in any of the animals of all dose groups.
Clinical findings noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be toxicologically relevant.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters were not affected by treatment.
Statistically significant variations noted in a few haematology parameters at 100 mg/kg were unrelated to treatment due to the slight magnitude of the difference from controls (values in treated rats remained within normal limits).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Lower total protein and albumin in males at all dose levels. The differences from controls increased with dose.
- Lower urea in males at 100 mg/kg.
- Higher total cholesterol in both sexes at 100 mg/kg.
The other statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the difference from controls (values in treated rats remained within normal limits) and/or absence of a dose-related response.
Thyroid hormone analyses:
Serum levels of T4, measured in F0 males, were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between treated and control animals.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted noted in the glandular stomach of both sexes and adrenal glands of females as described below.
Glandular stomach:
An increased incidence and severity of lymphogranulocytic inflammation was recorded in the glandular stomach of males and females starting at 10 mg/kg. This was accompanied by edema in some males at 100 mg/kg and females at 30 and 100 mg/kg.
Congestion/hemorrhage was recorded at an increased incidence and severity in males at 100 mg/kg.
The edema and congestion/haemorrhage of the glandular stomach recorded for females at 10 mg/kg, 30 mg/kg and for the control group were comparable in incidence and severity and therefore considered to be unrelated to the test item.
Adrenals:
In adrenal glands of females vacuolation of the zona glomerulosa, slightly above background, was recorded at 100 mg/kg.
The minimal vacuolation recorded for a single male at 10 mg/kg and a single female at 30 mg/kg was considered to be within background.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and normality of the estrus cycle were not affected by treatment.
Most females had regular cycles of 4 days. Extended di-estrus during pairing occurred in one female at 10 mg/kg (no. 55 which delivered no pups and had only one implantation; mating of this female was overlooked) and one female at 100 mg/kg (no. 76 which had total litter loss on Day 1 of lactation). The incidence of these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
spermatogenic staging profiles were normal for all males examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Mating index: Mating index was not affected by treatment. All females of all groups had mated.
- Fertility and Conception index: Fertility and conception index were not affected by treatment.
One control female (no. 44, mated with male no. 4) and one female at 100 mg/kg (no. 80, mated with male no. 40) were not pregnant (as confirmed by negative Salewski staining). No abnormalities were seen in the reproductive organs which could account for their nonpregnancy.
In the absence of a dose-related incidence, this non-pregnancy was considered not to be related to treatment.
- Precoital time: Precoital time was not affected by treatment.
- Number of implantation sites: Number of implantation sites was not affected by treatment.
For female nos. 59 (10 mg/kg), 62 (30 mg/kg) and 71 (100 mg/kg), the number of pups born was slightly higher than the number of implantations. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 16 of lactation.
- Microscopic examination of reproductive organs and spermatogenic profiling: There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.

Details on results (P0)

Developmental Data:
There was 1/10 couple (male 36 and female 76) at 100 mg/kg with total litter loss and 1/10 couple (male 15 and female 55) at 10 mg/kg without offspring. No abnormalities were seen in the reproductive organs which could account for their lack of healthy offspring. In the absence of a dose-related incidence, this lack of healthy) offspring was considered not to be related to treatment.

Gestation index and duration
Gestation index and duration of gestation were not affected by treatment.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-implantation survival index
Post-implantation survival index was not affected by treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Parental systemic
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOAEL
Remarks:
Parental local
Effect level:
< 10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

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):
No clinical signs occurred among pups that were considered to be related to treatment.
The nature and incidence of the clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not affected by treatment.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were not affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- Live birth index
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
The slightly lower mean number of living pups observed at first litter check at 100 mg/kg was caused by the total litter loss in a single female (no.76) within that group. An incidental total litter loss is a normal biological event and based on its single occurrence within this study considered on toxicological significance. Dead pups at first litter check were also observed in a few other females within this and other group(s). these number of dead pups were considered to be within the normal background incidence.

- Viability index
The number of live offspring on Day 4 before culling compared to the number of live offspring on Day 1 was not affected by treatment.
One pup at 10 mg/kg (litter no. 56) was killed in extremis on lactation Day 1 because this pups had a broken neck. Two pups of the control group (litters no. 45 and 47) and three pups at 10 mg/kg (litter no. 60) went missing on Days 2 or 3 of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

- Lactation index
No pups were found dead or went missing between lactation Days 5 and 13. For each group, the number of live offspring on Day 13 after littering was the same as the number of live offspring on Day 4 (after culling), resulting in a lactation index of 100% for all groups.

- Sex ratio
Sex ratio was not affected by treatment.

- Anogenital distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
In the absence of a dose-related response, the statistically significantly higher median absolute anogenital distance noted in female pups at 10 mg/kg was not related to treatment.

- Areola/nipple retention
Treatment up to 100 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Details on results (F1)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg).
No treatment-related or toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, viability and lactation indices, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, sex ratio, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
There were no treatment-related changes in the in-life parameters investigated (i.e. mortality, clinical appearance, functional observations, body weight and body weight gain, food consumption) and in the haematology parameters and organ weights in this study up to 100 mg/kg. No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg). No developmental toxicity was observed up to the highest dose level tested (100 mg/kg).
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Nocolok Cs Flux (SM) in rats by oral gavage was conducted according to the OECD guidelines 422 and 407 under GLP conditions.

Based on the results of a 28-day dose range finding study in which dose levels of 20, 100 and 500 mg/kg were tested and dose-limiting effects were noted at 500 mg/kg, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 10, 30 and 100 mg/kg.

The test item, formulated in water, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females with offspring were exposed for 50-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 42 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity and stability.

Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Parental results: There were no treatment-related changes the in-life results, haematology parameters or organ weights. Clinical biochemistry parameters showed treatment-related decreases in total protein and albumin, dose-dependently, in males at 10 mg/kg and above, a decrease in urea in males at 100 mg/kg, and an increase in total cholesterol in both sexes at 100 mg/kg. The changes at 100 mg/kg were considered to be toxicologically relevant. Microscopic examination revealed local treatment-related changes in the glandular stomach suggestive of irritating properties of the test item. These changes consisted of an increased incidence and severity of lymphogranulocytic inflammation in both sexes starting at 10 mg/kg, accompanied by edema in some males at 100 mg/kg and some females at 30 and 100 mg/kg. Additionally, males at 100 mg/kg showed congestion/haemorrhage which correlated with the macroscopically observed dark red/reddish foci in the glandular stomach of these males. Treatment-related vacuolation of the zona glomerulosa in the adrenal gland, slightly above background, was noted at 100 mg/kg in females. In the absence of any degenerative changes, this finding was considered to be non-adverse. No reproduction or developmental toxicity was observed up to the highest dose level tested (100 mg/kg). Based on the above results, the following No Observed Adverse Effect Levels (NOAELs) were derived.

- Parental systemic NOAEL: 30 mg/kg (based on changes in clinical biochemistry parameters at 100 mg/kg: lower total protein, albumin and urea, higher total cholesterol).

- Parental local NOAEL: < 10 mg/kg (based on histopathological changes in the glandular stomach at 10 mg/kg and above).

- Reproduction NOAEL: at least 100 mg/kg.

- Developmental NOAEL: at least 100 mg/kg.