Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
reproductive toxicity, other
Remarks:
Combined repeated dose toxicity study with the reproductive/developmental toxicity screening test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-21 to 2018-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2017-05-08
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10 °C to +25°C in a tightly closed original container, stored in a dry, cool and well venitlated place.
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at first administration: males: 74 days; females: 75 days
- Weight at first administration: males: 407.0 g - 477.5 g; females: 242.5 g - 278.0 g
- Housing (with the exception of the mating period): kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg)
- Diet (ad libitum): certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH); food residue was removed and weighed.
- Water (ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity:55 % ± 15 % (maximum range)
- Air changes: 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to provide dose concentrations of 37.5, 125 or 375/250 mg/mL
The test item formulations were freshly prepared every day and were adjusted to the animal's actual body weight daily.
The control animals received the vehicle at the same administration volume daily in the same way.

- Administration volume: 2 mL/kg bw/day


Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: the female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approx. 15 mL were taken at the following times and stored at ≤-20°C ± 10% until analysis:
1) At start of the treatment period (first dosing day):
- analysis of stability and concentration: immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature (3 samples / dose level group).
- homogeneity: immediately after preparation, during (middle) administration and before administration to the last animal/dose level group (3 samples / dose level group plus one sample/control).

2) At dose change (highest dose group, males)
- analysis of stability and concentration: immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature (3 samples of highest dose group).
- homogeneity: immediately after preparation, during (middle) administration and before administration to the last animal/dose level group (3 samples of highest dose group).

3) towards the end of the treatment period (when the majority of animals was dosed):
- analysis of concentration: during treatment always before administration to the last animal/dose level group (1 sample / group; low and intermediate dose groups; 2 samples from highest dose group: 1 sample from the male and 1 sample from the female group).
Duration of treatment / exposure:
- males: 35 treatment day (2 weeks prior to mating (from test day 15 until test day 29), during the mating period (14 days at maximum) and during the post-mating period until one day before sacrifice)
- females: 50 to 62 treatment days (2 weeks prior to mating, during the mating period (14 days at maximum) and during the lactation period until lactation day 13).
Frequency of treatment:
once daily
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
administered to both sexes
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
administered to both sexes
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
administered to both sexes, but due to the premature death of 2/10 males, the dose level was reduced from 750 to 500 mg/kg bw/day on test day 38 for the males.
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
starting on test day 38, this dose was administered to males instead of the 750 mg/kg bw/day dose due to premature deaths of the males in the high dose group.
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on available toxicological data and a 21-day dose range finding study (please refer to section 7.8.1 Toxicity to reproduction: s_Hansen_2018_DRF). In the 21-day dose range finding study, cesium tetrafluoroaluminate was administered orally to male and female rats at dose levels of 2, 10, 50, 100, 200, 600 or 1000 mg/kg bw/day for 3 weeks from test days 1 to 21.
At 1000 mg/kg bw/day one of 5 male animals died prematurely (found dead on the day of necropsy on test day 22) after 21 days of dosing. A post-dosing salivation (salivation started immediately to 5 minutes after dosing and disappeared again between 20 and 60 minutes after dosing) was noted from a dose level of 50 mg/kg bw/day onwards. The observation of salivation was related to the dose level (1 of 5 affected male animal at a dose level of 50 mg/kg bw/day and 5 of 5 affected male and female animals from 200 mg/kg bw/day onwards).
A reduced body weight and body weight gain was noted for the male animals at 1000 mg/kg bw/day but not for the female animals.
After 21 days of dosing statistically significant changes were noted for one or both sexes for several haematological parameters at 100, 200, 600 and 1000 mg/kg bw/day. In detail, slightly to moderately decreased levels were noted for the haemoglobin concentration, the number of red blood cells, the haematocrit value and the mean corpuscular volume.
A moderate to marked increase was noted for the number of white blood cells at dose levels of 600 and 1000 mg/kg bw/day.
The macroscopic examination at necropsy after 21 days of dosing revealed test item-related changes at 1000 mg/kg bw/day for the male animals in the form of small testes (1 of 5 males) and stomach changes (thickened mucosa, ulcera; 3 of 5 males).
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
clinical signs: before and after dosing at each time of dosing as well as regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approx. 3:30 p.m.
mortality: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays, a similar procedure was followed with a final check at approx. 3.30 p.m.
- Cage side observations checked: behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity as well as mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) and once a week thereafter (performed at least 2 hours after dosing)

BODY WEIGHT: Yes
- Time schedule for examinations:
1) Males: weekly starting during the premating period and at sacrifice

2) Females:
premating period: weekly
gestation period: gestation days 0, 7, 14 and 20
lactation period: lactation days 1,4 and 13

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg bw/day) was determined using the following
formula: Relative food consumption = (total food given (g) - total food left (g))/number of animal days* x body weight (kg)
*the term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

Food residue (or total food left) was weighed and recorded as followed:
- Pre-mating period
males and females: TD8 and TD15
- Mating period:
males and females: none
- Gestation period
males: not applicable
females: GD7, GD14 and GD21
- Lactation period
males: not applicable
females: LD1, LD8 and LD12

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual appraisal throughout the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
at the end of the premating period on test day 29
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals:
5 animals/sex/group
- Parameters checked:
haemoglobin content, erythrocytes, leucocytes, differential blood count (relative and absolute; neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes as well as large unstained cells), reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thromboplastin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
at the end of the premating period on test day 29
- Animals fasted: Yes, overnight
- How many animals:
5 animals/sex/group
- Parameters checked:
albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), blood urea, calcium, chloride, potassium, sodium, sodium/potassium ratio, blood urea/creatinine ratio, lactate dehydrogenase, alanine aminotransferase, alkaline phosphatase and aspartate aminotransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
at least two hours after dosing and before any blood sampling for laboratory examinations:
males: test day 48
females: test day 64, 65, 66, 70 or 73
- Dose groups that were examined:
all dose groupy (5 animals/sex/dose)
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

THYROID HORMONE (T4) DETERMINATION: Yes
- Time schedule for examinations: at scheduled sacrifice (always at the same time in the morning)
- Animals fasted: Yes, overnight
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- How many animals: all parental males

FURTHER OBSERVATIONS:
- duration of pre-coital time
- gestation length
- number of implantation sites
Oestrous cyclicity (parental animals):
During the 14-day pre-exposure period (TD 1 to TD 14), the oestrus cycle of the female animals was monitored to yield study groups of at least 10 animals each with a normal oestrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded from the randomization process on test day 14 that was performed to allocate the female animals to the test groups of the main study. Most of the female animals that were used for the main study revealed 2 or 3 typical 4 to
5 day cycles during the 14-day pre-exposure period.
After the allocation of the animals to the study groups and the start of treatment, the oestrus cycle was further monitored during the pre-mating period and the
mating period until one day before a positive mating sign was noted.
Finally, a vaginal smear was taken in the morning of the day of scheduled necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight, qualitative stages of spermatogenesis and testicular structure

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, the litters were adjusted to 10 pups/litter by eliminating (culling) surplus pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring as soon as possible after delivery: number and sex of pups, stillbirths, live births, runts, presence of gross anomalies and behavioural abnormalities
Live pups were counted, sexed and weighed on post-natal days 1, 4 and13.
On post-natal day 4 before litter adjustment the ano-genital distance of all pups was determined usng a scale.
Nipples were counted in all male pups on post-natal day 13 (shortly before scheduled sacrifice).
Clinical signs were observed daily.

THYROID HORMONE (T4) DETERMINATION
- Time schedule for examinations: postnatal day 13
- Animals fasted: No
- How many animals: at least 2 pups/litter (if possible from one male and one female; all litters
Postmortem examinations (parental animals):
GROSS PATHOLOGY:
The animals were sacrificed at the following times:
- males: on test day 50
- dams (surviving dams): on lactation day 14
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
During necropsy the number of implantation sites was recorded in the female animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

ORGAN WEIGHTS.
The weight of the following organs of all adult male and female animals was determined before fixation (where applicable): adrenal glands, brain, epididymis, heart, kidneys, liver, ovaries, spleen, testicles, thyroid, thymus, prostate, seminal vesicles with coagulating glands, uterus including cervix

HISTOPATHOLOGY:
The following organ(s) or parts thereof of all adult male and female animals were fixed in modified Davidson's solution or 7% buffered formalin: epididymis, testicles, gross lesions observed, ovaries and oviducts, prostate, seminal vesicles with coagulating glands, thyroid (including parathyroids), uterus (including cervix) and vagina.
Any other organs displaying macroscopic changes were also preserved.
5 male and female animals from each group were selected for histopathology examination and the following organs from the selected animals and from every deceased or prematurely sacrificed animal were fixed for histopathology examination: eye with optic nerve (2), epididymis (2), testicle (2), adrenal gland (2), bone, bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem (pons)), gross lesions observed, heart (3 levels: right and left ventricle, septum), intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), intestine, large (colon, rectum), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles), preserved by inflation with fixative and then immersion, lymph node (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal), nerve (sciatic), oesophagus, ovary and oviduct, pituitary, prostate and seminal vesicles with coagulating glands, spinal cord (3 sections), spleen, stomach, thyroid (including parathyroids), thymus, tissue masses or tumors including regional lymph nodes), tongue (including base), trachea (including larynx), urinary bladder, uterus (including cervix) and vagina.

Full histopathology was performed on the preserved organs of the selected parental animals of the control group and the highest dose group. The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of the control group and the highest dose group following H-E and PAS staining.
Postmortem examinations (offspring):
SACRIFICE
The pups were sacrificed on postnatal day 13.

GROSS NECROPSY
Dead pups and pups sacrificed at day 13 post-partum were examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid of 1 male and 1 female pup from each litter was fixed in 7% formalin.
Thyroid weight was determined after fixation.
The thyroid of selected pups was histopatholoically examined.
Statistics:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant
effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi² test.
Reproductive indices:
- female fertility index [%] = (number of pregnant rats / number of rats used) x 100
The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed
litters.
- gestation Index [%] = (number of dams with live pups / number of pregnant rats) x 100
- birth Index [%] = (total number of pups born (alive + dead) / number of implantation scars) x 100
- post-implantation loss [%] = (implantations - living fetuses / implantations) x 100
Offspring viability indices:
- live birth index [%] = (number of pups alive on day 0/1 of lactation / total number of pups (alive + dead)) x 100
- viability index [%] (pre-cull) = (number of pups alive on day 4 (pre cull) / number of pups alive on day 0/1) x 100
- viability index [%] (post cull) = (number of pups alive on day 13 / number of pups alive on day 4 (post cull)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs:
1) Males:
- 750/500 mg/kg bw/day: a haemorrhagic nose/snout for 3/8 surviving animals was observed on several test days (considered to be test item-related).

2) Females:
-750 mg/kg bw/day: piloerection was noted for all 4 surviving females during the lactation period on altogether 36 test days (considered to be test item-related).
- premature deaths (750 mg/kg bw/day): the observation of piloerection that was noted for 5 of the 6 prematurely deceased animals was also noted for all 4 surviving animals and therefore considered to be test item-related and not as a pre-mortal symptom
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
1) Males:
750/500 mg/kg bw/day: 2/10 male animals died prematurely, which was considered to be test item-related due to changes in the laboratory parameters, reductions in body weight and changes in organ weight.

2) Females:
750 mg/kg bw/day: 6/10 females died during the lactation period between test days 54 to 64 (corresponding to lactation days 1 to 9). Due to the high incidence of prematurely deceased high dosed females, these premature deaths were considered to be test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 750/500 mg/kg bw/day: a statistically significantly reduced body weight in comparison to the control group was noted from test day 29 until sacrifice on test day 50 (considered to be test item-related). In detail, on test day 29 the body weight of the males was 7.7 % below the value of the control group (p ≤ 0.05). Thereafter the difference in body weight slightly increased. On test day 49 (last day of dosing) the body weight of the animals was 9.5% below the value of the control group (p ≤ 0.01) and at sacrifice (test day 50) the body weight of the male animals was 11.0 % below the control value (p ≤ 0.01). The percentage of body weight gain was decreased in the high dose group in comparison to the control group and the 75 and the 250 mg/kg bw/day dose groups.
The body weight at autopsy for the 8 surviving male animals of the 750/500 mg test item/kg bw/day dose group was 10.7 % below the value of the control group (p ≤ 0.01). This was in the range of the difference that was noted for the live animals on the day of sacrifice (11.0 % below the control value) and considered to be test item-related.
- premature death (750/500 mg/kg bw/day): one male showed a marked reduction in body weight before death (minus 30.6 % between test day 15 and test day 29

2) Females:
- 750 mg/kg bw/day: a statistically significantly (p ≤ 0.05 or 0.01) reduced body weight was noted from gestation day 14 onwards until the end of the lactation period. This reduction in body weight in comparison to the control group was considered to be test item-related. In detail, the mean body weight of the females was 7.5 % below the control value on gestation day 14 (p ≤ 0.05). At the beginning of the lactation period (lactation day 1) the mean body weight of the 9 living females (one female died during littering on lactation day 1) was 14.4 % below the control value (p ≤ 0.01). During the further course of the lactation period 5 further females of the treatment group died and the mean body weight of the 4 surviving animals on lactation day 13 was 21.9 % below the value of the control group (p ≤ 0.01). In accordance with the reduced body weights that were noted during the gestation and the lactation period, body weight gain for these periods was reduced at the high dose level in comparison to the control group.
A slight and statistically not significant reduction in body weight at autopsy (9.6% below the value of the control group) was noted for the 4 surviving female animals on lactation day 14. This was in agreement with the test item-related reduction in live body weight that was noted on lactation day 13 (21.9 % below the control value).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1) Females:
- 750 mg/kg bw/day: during the second week of the lactation period a reduction in food consumption (54.0% below the value of the control group, p ≤ 0.01) was noted for the 4 surviving female animals of the treatment group. This may be caused by the damaged teeth that were noted for the 4 surviving females at necropsy and/or the poor health condition of these 4 animals in general, as demonstrated by the reduced body weight or the poor results of the neurological screening. Due to these reasons, the reduced food consumption of the 4 surviving animals during the second week of the lactation period is considered to be test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
Note: The 5 selected males of the 750/500 mg/kg bw/day dose group that were selected for the laboratory examinations included the 2 prematurely deceased animals).
- 250 mg/kg bw/day: a slight and statistically not significant increase was noted for the number of white blood cells (47.5 % above the value of the control group). Regarding the subunits of the white blood cells, statistically significant increases were noted for the numbers of neutrophilic and eosinophilic granulocytes (p ≤ 0.01 and p ≤ 0.05, respectively). The increases in the number of neutrophilic and eosinophilic granulocytes were considered to be test item-related, as more pronounced increases were noted at the750/500 mg/kg bw/day dose level (dose-response relationship).
- 750/500 mg/kg bw/day: the increase in the number of white blood cells was more pronounced as at the 250 mg/kg bw/day dose level and statistically significant (143.3 % above the value of the control group; p ≤ 0.05). The cell numbers of nearly all subunits of the white blood cells (cell numbers of lymphocytes, monocytes, neutrophilic, eosinophilic and basophilic granulocytes, with the exception of the small population of large unstained cells) were markedly increased (statistically significant or not; p ≤ 0.01 or 0.05)). All changes that were noted at the 750/500 mg/kg bw/day dose level for the above mentioned parameters were considered to be test item-related. However, it has to be mentioned that a high variability was noted for the above mentioned parameters at the 750/500 mg test item/kg bw/day) dose level. Markedly increased numbers of white blood cells, lymphocytes, monocytes, neutropholic, eosinophilic and basophilic granulocytes were especially noted for the 2 prematurely deceased animals (one animal died 1 day after blood withdrawal on test day 30 and one animal died 7 days after blood withdrawal on test day 36) and one surviving animal. In contrast, the values of the above mentioned parameters for other two surviving animals were in the range of the control group (only for the number of neutrophilic granulocytes an increased value was noted for one animal).

2) Females:
Note: The 5 females of the 750 mg/kg bw/day dose level that were selected for the laboratory examinations included the 4 surviving females and one prematurely deceased female.
- 250 and 750 mg/kg bw/day: increased numbers of neutrophilic granulocytes, eosinophilic granulocytes and monocytes were noted at the 250 and the 750 mg/kg bw/day dose levels (considered to be test item-related). The increases in the above mentioned cell numbers were statistically significant at p ≤ 0.05 / 0.01 or not statistically significant in comparison to the control group and slightly more pronounced at the 750 mg/kg bw/day dose level as at the 250 mg/kg bw/day dose level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 250 mg/kg bw/day: a slight but statistically significantly decreased potassium concentration was noted (p ≤ 0.01). Though all individual values were clearly within the laboratory background data, they were all slightly below the individual values of the control group. This small reduction was considered to be test item-related due to the test item-related reductions that were noted for the potassium concentrations at the 750/500 mg/kg bw/day dose levels of the male and more pronounced of the female animals.
- 750/500 mg/kg bw/day: test item-related changes were noted for the potassium concentration, the aspartate aminotransferase activity, the albumin and globulin concentration and the urea in blood concentration. The findings are described in detail below.
A statistically significantly decrease in potassium concentration was noted in males (p ≤ 0.01). A marked decrease in the potassium concentration was noted for one surviving male animal and one prematurely deceased animal (values below or at the lower range of laboratory background data), whereas the potassium concentrations of the remaining 3 animals remained at the control level. Though only 2 of 5 animals showed a decreased potassium concentration at the high dose group, this was considered to be test item-related, as a decreased potassium level was also noted for the female animals of the high dose group.
Furthermore, a statistically significantly increase in aspartate aminotransferase activity was observed at the high dose group (p ≤ 0.01). An increased aspartate aminotransferase activity that was above the upper range of the background data was noted for 4 of the 5 examined male animals. of the high dose group.
A slight but statistically significant decrease was noted for the protein (total) concentration and thus for the albumin and globulin concentrations (p ≤ 0.05 or p ≤ 0.01). All individual values for protein (total) and globulin were within the laboratory background data, whereas two individual values for albumin were slightly below the background data.
A moderately but statistically not significant increase was noted for the urea in blood concentration. Though this increase was mainly due to a markedly increased level of one prematurely deceased animal, it was considered to be test item-related as the urea in blood concentration of the high dosed females was also increased.

2) Females:
- 750 mg/kg bw/day: test item-related changes were noted for the electrolyte levels (potassium, chloride and calcium concentration), the urea in blood concentration, the cholesterol concentration and the aspartate aminotransferase activity. In detail, a statistically significantly decrease was noted for the potassium and chloride concentration and a statistically significantly increase for the calcium concentration (p ≤ 0.05 or p ≤ 0.01). Especially the effect on the potassium concentration was highly significant. All individual values were markedly below the lowest value of the other test groups (control, 75 and 250 mg/kg bw/day group) and at the lower range of thelaboratory background data.
A clear effect was also noted for the chloride concentration. Though the difference was rather small (3.3 % below the control value; p ≤ 0.01), 4 of 5 individual values of the high dose group were below the lower value of the control group (only the value of one 750 mg/kg bw/day dose group female was at the same level as the lowest value of the control group). Furthermore, all values were below or at the lower range of background data.
A statistically significantly increase was noted for aspartate aminotransferase activity (p ≤ 0.01). However, a markedly increased aspartate aminotransferase activity was only noted for one female, whereas the aspartate aminotransferase activities of the other high dosed females were only slightly increased, but within the laboratory background data. However, due to the more pronounced increased aspartate aminotransferase activity that was noted for the male animals, this was considered to be test item-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
1) Females:
- 750 mg/kg bw/day: an adverse effect was noted on some of the examined motoric skills (limb rotation, wire maneuver, positional passivity, positive geotropism, righting reflex) for the 4 surviving female animals that were used for the observational screening tests (considered to be test item-related). In addition, a statistically significantly reduced fore- and hindlimb grip strength was noted for the 4 surviving females of the 750 mg test item/kg bw/day dose group (p ≤ 0.01 and p ≤ 0.05), respectively). The grip strength values of the individual animals were below or near the lower range of laboratory background data. The decreased grip strength was considered to be test item-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 750/500 mg/kg bw/day:
Adrenals:
- a slight to marked hypertrophy of the cortex was noted for 3 of 5 male animals (one suriving male and the two prematurely deceased animals; considdered to be test item-related).

Heart:
- a slight to moderate necrosis/inflammation was noted for 3 of 5 male animals (one surviving animal and the two prematurely deceased animals; considered to be test item-related).

Kidneys:
- several changes were noted for all examined animals (e.g. basophilic tubules in the cortex, degeneration of the proximal and distal tubules, glomerulonephritis, oil red staining; considered to be test item-related).

Lymph node (mesenteric):
- a sinus histocytosis was noted for 2 of 5 animals (one surviving animal and one prematurely deceased animal; considered to be test item-related).

Skeletal muscle:
- a muscle degeneration together with a granulomatous inflammation was noted for 2 of 5 male animals (one surviving male and one prematurely deceased animal; considered to be test item-related).

Stomach:
- several findings were noted for altogether 4 of 5 dosed animals (e.g. acute inflammation of the mucosa/submucosa, ulcertion of the mucosa; considered to be test item-related).

Thymus:
- a marked to severe atrophy was noted for 4 of 5 dosed males (three surviving animals and one prematurely deceased animal; considered to be test item-related).

Testes:
- two males of the 750/500 mg/kg bw/day dose group revealed degenerated/atrophied and dilated left and right testes during the microscopic examination. In case of one male the microscopic testes findings were correlated with macroscopic findings in the form of an enlarged and malpositioned left and right testis.

2) Females:
Note: all 10 females of the 750 mg/kg bw/day dose group were examined histopathologically: 6 prematurely deceased females and the 4 surviving females. The 4 surviving females were included in the 5 selected 750 mg/kg bw/day dose females that were selected for the histopathological examination at the begin of the study.

- 750 mg/kg bw/day:
Adrenal gland:
- a slight hypertrophy of the cortex was noted for 2/10 female animals (two prematurely deceased animals; considered to be test item-related).

Heart:
- a minimal or moderate necrosis/inflammation was noted for 4 of 10 female animals (prematurely deceased females; considered to be test item-related).

Kidneys:
- several changes were noted for 8/10 examined females (e.g. basophilic tubules in the cortex, degeneration of the proximal and distal tubules, oil red staining). With the exception of oil red staining (7 of 10), all other kidney observations were only noted for 1 or 2 different females of the high dose group. Findings were considered test item-related.

Lungs:
- Granulomas were noted for 2 of 10 animals (considered to be test item-related)

Lymph nodes:
- a sinus histocytosis was noted in the mesenteric lymph node from 7/10 females. Also 7/10 females revealed a plasmacytosis in the cervical lymph node. Findings were considered test item-related.

Skeletal muscle:
- a muscle degeneration together with a granulomatous inflammation was noted for 5 of 10 female animals (considered to be test item-related)

Spleen:
- an atrophy of the white pulp was noted for 5 of 10 females (considered to be test item-related).

Stomach:
- several findings were noted for altogether 9 of 10 females (e.g. erosions of the glandular mucosa, acute inflammation of the mucosa/submucosa, ulcertion of the mucosa; considered to be test item-related).

Thymus:
- a marked to severe atrophy was noted for all (10 of 10) females (considered to be test item-related).

Urinary bladder:
- a hyperplasia of the urothelial cells was noted for 3 of 10 females (considered to be test item-related).

Uterus:
- changes were noted in the uterus from 3 of 10 females in the form of an inflammation and / or an haemorrhagic endometrium. A slight to moderate mucification of the vaginal mucosa was noted for 4 of 10 females. These findings are considered to be test item-related.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
- 750/500 mg/kg bw/day: a highly significant effect was noted at the dose level in the form of increased organ weights of the testes (absolute and relative), which was correlated with the macroscopic finding of enlarged testes at necropsy. In detail, 7/8 surviving male animals of the 750/500 mg/kg bw/day dose group revealed a markedly increased relative and absolute organ weight of the left testis in comparison to the control group. This was correlated with the macroscopic finding of an enlarged left testis in 4 high dosed animals at necropsy. Increased relative and absolute organ weights were also noted for the right testes (statistically not significant), which was correlated with the macroscopic finding of an enlarged right testis in 2 high dosed males. However, 2 animals were noted with macroscopically small right testis. The relative and the absolute organ weights of the right testes of these 2 animals were below the lowest value of the other test groups (control, 75 and 250 mg/kg bw/day dose group). Both, the increased and the decreased testes weights were considered to be test item-related.
Two males of the 750/500 mg/kg bw/day dose group revealed degenerated/atrophied and dilated left and right testes during the microscopic examination. In case of one male the microscopic testes findings were correlated with macroscopic findings in the form of an enlarged and malpositioned left and right testis.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- 750 mg/kg bw/day: 1/9 pregnant females (animal which died during littering was excluded) delivered only stillbirth, leading to a reduced gestation index of 89 %. Such a single occurrence is in the spontaneous range. However, due to the marked adverse effects that were noted for the prenatal development of the pups at the 750 mg/kg bw/day dose level, the reduced gestation index as well as the delivering of only stillbirths by the one animal has to be considered as test item-related.
Furthermore, a statistically significantly (p ≤ 0.01) elongated gestation length was noted at the 750 mg/kg bw/day dose level. This was considered to be test item-related. In detail, the females showed a gestation length of 23 or 24 test days, whereas in the control group gestation lengths were noted between 21 and 23 test days. The mean gestation length at the 750 mg/kg bw/day dose level was 23.3 test days and above the range of the laboratory background data.

- 750 mg/kg bw/day: the mean number of implantation sites per dam was statistically significantly (p ≤ 0.01) reduced by 25.8% (11.8 implantation sites per dam at the high dose level in comparison to 15.9 implantation sites per dam from the control group). Additionally to the reduced number of implantation sites, an adverse effect of the test item was also noted on the development of the implanted eggs (demonstrated by an increased number of resorptions and a high number of stillbirths). This led to a reduced birth index and a reduced live birth index at the high dose level.
4.9 implantations per dam of the high dose group got lost as a resorption or a stillborn on average, in comparison to only 1.1 lost implantations per dam from the control group. The increased number of resorptions and stillbirths led to a markedly increased post-implantation loss at the high dose level (41.5 % in comparison to only 7.1 % in the control group on the group level).
CLINICAL SIGNS/DETAILED CLINICAL SIGNS:
Clinical signs:
1) Males:
- 0 mg/kg bw/day: no changes in behaviour, the external appearance or the appearance of the faeces were noted.
- 75 mg/kg bw/day: no changes in behaviour, the external appearance or the faeces were noted that were of toxicological relevance. However, a slight salivation was noted for 2/9 animals on one day each. Such a post-dosing salivation that started briefly after dosing and lasted for a short time is often observed in gavage studies and maybe a reaction to the taste or irritation of the test article, rather than an indication of toxicity
- 250 mg/kg bw/day: salivation was observed in 6/10 animals (not toxicologically relevant). Furthermore, haemorrhagic urine was noted for one animal on 4 consecutive test days. As only 1 animal was affected this observation was considered to be spontaneous.
- 750/500 mg/kg bw/day: salivation was noted for all 8 surviving animals (not toxicologically relevant).
- premature death (75 mg/kg bw/day; one male): the observations that were noted a few days before death during the daily cage side observations (e.g. prone position) were considered to be caused by the misgavage and the following poor health state of the animal.

2) Females:
- 0 mg/kg bw/day: no changes in behaviour, the external appearance or the appearance of the faeces were noted.
- 75 and 250 mg/kg bw/day: no observations were noted that were considered to be of toxicological relevance. In the low dose group, slight to moderate salivation was observed in 2/9 animals during the gestation period. In the 250 mg/kg bw/day dose group, slight to pronounced salivation was observed in 1/10, 7/9 and 4/9 animals during the premating/mating, gestation and lactation period, respectively.
- 750 mg/kg bw/day: salivation was noted in 5/10, 9/10 and 2/4 animals during the premating/mating, gestation and lactation period, respectively (not toxicologically relevant).
- premature deaths (750 mg/kg bw/day): pre-mortal symptoms were noted during the daily cage side observations for all prematurely deceased animals in the form of convulsions, tremors, lateral position, reduced motility, breathing sounds and or a haemorrhagic nose/snout.

Start and duration of observations:
- salivation: with only a few exceptions, salivation started immediately to 5 min after administration and disappeared again between 20 and 60 min after administration.
- reduced motility: a reduced motility was only recorded for 2 animals on their day of death (premature death (one male and one female; high dose group).
For the male animal a reduced motility was soon noted in the morning at work begin on test day 30 and lasted until its premature death on test day 30. Due to the poor health condition of the male no administration of test item was carried out anymore on this day. Hence, no correlation of the symptoms to the time point of administration was possible on this day. In case of the female animal a reduced motility was noted immediately to 5 min after administration in the morning of lactation day 9 and persisted until its premature death during the further course of lactation day 9.
- prone position: prone position was noted for the prematurely deceased male of the 75 mg/kg bw/day dose group on test day 47 and on test day 48. It started between 20 and 60 min after administration on test day 47 and persisted until the premature death on the following day (test day 48).
- convulsions: convulsions were noted for 4 of 6 prematurely deceased high dosed females one day before and/or on their day of death. It started immediately to 2 hours after administration and disappeared between 5 min and 2 hours after administration.
- tremors: tremors were noted for prematurely deceased female of the 750 mg/kg bw/day dose group, on lactation day 1, one day before the animal was found dead on lactation day 2. Tremors were noted immediately to 5 min after administration and disappeared between 20 and 60 min after administration.


Detailed clinical signs:
1) Males:
- 0, 75, 250 and 750/500 mg/kg bw/day: no external observations, no changes in body posture, movement and coordination and in behaviour were noted for the male animals of the treatment groups and the control group.

2) Females:
- 0, 75, 250 and 750 mg/kg bw/day: no external observations, no changes in body posture, movement and coordination and in behaviour were noted for the female animals of the treatment groups and the control group.

MORTALITY:
1) Males:
- 0, 75 and 250 mg/kg bw/day: no test item-related death was noted. One male of the 75 mg/kg bw/day dose group died during test day 48 (two days before
scheduled removal on test day 50). The premature death of the animal was considered to be caused by a misgavage a few days before death and not test item-related.

2) Females:
- 0, 75 and 250 mg/kg bw/day: no test item-related premature death was noted.

BODY WEIGHT AND WEIGHT CHANGES:
1) Males:
- 75 and 250 mg/kg bw/day: no test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the dose groups. Furthermore, no test item-related differences were noted on the body weight at autopsy between the control group and the treatment groups.

2) Females:
- 75 and 250 mg/kg bw/day: no test item-related differences in body weight and body weight gain were noted between the female rats of the control group and the female rats of the treatment groups during the premating, the gestation and the lactation period. Furthermore, no test item-related differences were noted on the body weight at autopsy between the control group and the treamtent groups.

FOOD CONSUMPTION:
1) Males (Pre-mating period):
- 75, 250 and 750/500 mg/kg bw/day: during the pre-mating period (test days 15 to 28) no test item-related difference in food consumption was noted between the rats of the control group and the rats of the treatment groups.

2) Females (Pre-mating, gestation and lactation period):
- 75 and 250 mg/kg bw/day: no test item-related differences in food consumption were noted between the female rats of the control group and the female rats of the treatment groups during the premating, the gestation and the lactation period.
- 750 mg/kg bw/day: no test item-related differences in food consumption were noted during the pre-mating period, the gestation period and the first week of the lactation period (lactation days 1 to 8).

DRINKING WATER CONSUMPTION:
Males and females:
- 75, 250 and 750/500 mg/kg bw/day: no test item-related changes in the consumption of drinking water was noted for the male and female rats.

HAEMATOLOGICAL FINDINGS:
1) Males.
Note: The 5 selected males of the 750/500 mg/kg bw/day dose group that were selected for the laboratory examinations included the 2 prematurely deceased animals).
- 75 mg/kg bw/day: no test item-related influence on the examined haematological parameters were noted.
- 250 and 750/500 mg/kg bw/day: slightly decreased haemoglobin contents were noted (statistically significant at the 250 mg/kg bw/day dose level, p ≤ 0.05). However, as the changes were only slight and showed no dose-response relationship, they were considered to be spontaneous.
- 75 and 750/500 mg/kg bw/day: slightly decreased MCH and MCV values were noted (statistically significant for the MCH value at the 75 mg/kg bw/day dose level (p ≤ 0.05) and for the MCH value and the MCV value at the 750/500 mg/kg bw/day dose level (p ≤ 0.01)). However, no decreased MCH and MCV values were noted at the 250 mg/kg bw/day dose level. As the MCH and MCV values at the 250 mg/kg bw/day dose level were at the level of the control group, a dose-response relationship from the 75 mg/kg bw/day to the 750/500 mg/kg bw/day dose level was missing. Hence, the changes that were noted for the MCH and MCV values were considered to be spontaneous.

2) Females:
Note: The 5 females of the 750 mg/kg bw/day dose level that were selected for the laboratory examinations included the 4 surviving females and one prematurely deceased female.
- 75 mg/kg bw/day: no test item-related influence on the examined haematological parameters were noted.
- 250 mg/kg bw/day: a slight but statistically significantly decrease of activated partial thromboplastin time (p ≤ 0.01) was noted, whereas the activated partial thromboplastin time at the 750 mg/kg bw/day dose level was again in the range of the control group. As no dose-response relationship was noted, this observation was considered to be spontaneous.

CLINICAL BIOCHEMISTRY FINDINGS:
1) Males:
- 75 mg/kg bw/day: no test item-related changes for the examined biochemical parameters were noted.

2) Females:
- 75 and 250 mg/kg bw/day: no test item-related changes for the examined biochemical parameters were noted.
- 250 and 750 mg/kg bw/day: the slightly statistically significantly decreased protein (total) and globulin concentrations that were noted at the 250 and the 750 mg/kg bw/day dose group were considered to be spontaneous. All values were in the range of the mean value of the laboratory background data. The significant differences between the control group and the 250 and the 750 mg/kg bw/day dose group may be due to an increased protein (total) concentration at the control group. This assessment is in contrast to those that was made for the male animals for these parameters. However, whereas all protein (total) values from the 750 mg/kg bw/day dosed females were near the mean value of the laboratory background data, a higher variability was noted for the male animals and 2 of 5 values from the 750/500 mg/kg bw/day dosed males were below the lower range of the background data.
- 75, 250 and 750 mg/kg bw/day: the statistically significantly decreased glucose concentrations that were noted were due to an increased glucose concentration at the control group and considered to be spontaneous. All mean values of the treatment groups were at the same level and in the range of the mean value of the laboratory background data.
- 250 and 750 mg/kg bw/day: slightly decreased alanine aminotransferase activities were noted (statistically significant or not). However, increased activities are an indicator for organ damage (alanine aminotransferase release from destroyed cells), whereas decreased activities were not of toxicological relevance and could be considered as spontaneous.

NEUROBEHAVIOURAL EXAMINATION:
1) Males:
- 75, 250 and 750/500 mg/kg bw/day: no test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faeces were noted for the male animals of all treatment groups. Furthermore, no test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group. Also, no test item-related influence on the fore- and hindlimb grip strength was noted for the treated male animals. Lastly, no test item-related differences in spontaneous motility were noted between the control group and the treatment groups. However, statistically significantly reduced numbers of movements were noted at the low dose level (57.1 % below the control value; p ≤ 0.05) and the high dose level (70.2 % below the control value; p ≤ 0.01). These observations were considered to be spontaneous and not test item-related due to the following reasons: a high standard deviation was noted for all groups and nearly all individual values were in the range of laboratory background data (only one animal of the high dose group with only 26 movements in 12 minutes was slightly below the lower laboratory background range).

2) Females:
Note: at the 750 mg/kg bw/day dose level only 4 surviving female animals were still available at the time point of the neurological screening.
- 75 and 250 mg/kg bw/day: no test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faeces were noted. Furthermore, no test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group. Also, no test item-related influence on the fore- and hindlimb grip strength was noted for the female animals of the low and the intermediate dose group.
- 75 and 750 mg/kg bw/day: the slight but statistically significantly increased hind leg splay that was noted for the animals of the 75 and the 750 mg/kg bw/day dose level (13.4 % and 17.4 % above the control group; p ≤ 0.05) was not considered to be test item related. No dose response relationship was noted and all individual values were in the range of laboratory background data or only slightly above (one animal of the low dose group with a hindleg splay of 10.5 cm).
- 75, 250 and 750 mg/kg bw/day: no test item-related differences in spontaneous motility were noted between the control group and the treatment groups.

ORGAN WEIGHTS INCLUDING ORGAN7BODY WEIGHT RATIOS:
1) Males:
- 75 mg/kg bw/day: no test item-related changes were noted. The statistically significantly increased relative organ weight of the left kidney (p ≤ 0.05) that was noted revealed no dose-response relationship and was considered to be spontaneous.
- 250 mg/kg bw/day: slight, but statistically significantly decreased organ weights were noted for the absolute organ weights of the left and right epididimydes (p ≤ 0.01 and p ≤ 0.05, respectively). As the changes were only small and no statistically significant changes were noted for the relative organ weights of the left and right epididymis, these changes were considered to be spontaneous.
- 750/500 mg/kg bw/day: more pronounced changes as at the 250 mg/kg bw/day dose level (statistically significant decreased; at p ≤ 0.01) were noted for the absolute organ weights of the left and right epididymis, the combined prostate and seminal vesicle and the liver. However, these changes were considered to be due to the reduced body weight of the 750/500 mg/kg bw/day dosed animals, as no statistically significant changes were noted for the relative organ weights of these organs .
- 750/500 mg/kg bw/day: the slight but statistically significantly increased relative organ weight of the brain (p ≤ 0.01) that was noted was also due to the reduced body weight at the dose level, as the absolute organ weight of the brain was in the range of the control group.

2) Females:
- 75 mg/kg bw/day: no test item-related influence on the relative and the absolute organ weights was noted.
- 750 mg/kg bw/day: the statistically significantly decreased absolute organ weights of the heart and the liver (p ≤ 0.01) were considered to be due to the reduced body weight of the 4 surviving female animals, as no statistically significant decrease was noted for the relative organ weights.

GROSS PATHOLOGICAL FINDINGS:
1) Males:
- 75 and 250 mg/kg bw/day: no test item-related observations were noted for the surviving male animals. However, different observations were noted at the two dose level. These observations were considered to be spontaneous, as they were only noted for 1 (e.g. soft and enlarged left kidney) or 2 animals (observed for 2 animals: an urinary bladder which was filled with content, noted in animals of the low dose group). However, the urinary bladder and the kidneys were considered as target organs during the histopathological examination of the animals of 750/500 dose group.
- premature death (75 mg/kg bw/day; one male): the macroscopic findings at necropsy were considered to be due to the misgavage (abdomen filled with clear liquid or considered to be spontaneous (prostate findings). The observation of soft kidneys was correlated with microscopic kidney findings (e.g. basophilic tubules in the cortex) which were noted for the animal.

2) Females:
- 75 and 250 mg/kg bw/day: no test item-related observations were noted for the female animals. The stomach changes in the form of haemorrhagic foci that were noted for 1 female of the 75 mg/kg bw/day dose group and 1 female animal of the 250 mg/kg bw/day dose group were considered to be spontaneous, as only 1 animal per group was affected. However, due to microscopic and related macroscopic findings at the 750 mg/kg bw/day dose level, the stomach was considered as a target organ.

REPRODUCTIVE FUNCTION. OESTRUS CYCLE
- 75, 250 and 750 mg/kg bw/day: no differences were noted in the mean number of oestrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the treatment groups. However, several females with an elongated diestrus stage (between 9 to 16 consecutive test days) were noted before the day of mating. In detail, 2 animals with an elongated diestrus stage were noted in the control group, 4 in the low dose group, 1 in the intermediate dose group and 3 in the high dose group. As 2 animals with an elongated diestrus stage before mating were noted in the control group, the respective findings in the treatment groups were considered as spontaneous.

REPRODUCTIVE PERFORMANCE:
- 75, 250 and 750 mg/kg bw/day: no test item-related influence on the fertility index of the female rats was noted for any of the treatment groups. All female rats were successfully mated (confirmed by sperm detection). However, 2 female animals of the control group and one female animal of the 75 mg/kg bw/day and the 250 mg/kg bw/day dose group did not become pregnant, leading to a fertility index of 80% for the female animals of the control group and 90% for the animals of the 75 mg/kg bw/day and the 250 mg/kg bw/day dose group. The occurrence of one non-pregnant female each at the low dose level and at the intermediate dose level was considered as spontaneous, as even 2 non-pregnant females were noted at the control group.
- 75 and 250 mg/kg bw/day: no test item-related influence on the gestation index was noted for the female rats.
- 75, 250 and 750 mg/kg bw/day: no test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups. Conspicuously elongated pre-coital times of 12 test days were noted for one pregnant animal of the 75 mg/kg bw/day dose group and one pregnant animal of the 250 mg/kg bw/day dose group. These single occurrences were considered to be spontaneous. The pre-coital times of the pregnant 750 mg/kg bw/day dosed females (1 to 5 test days) were in the range of the pre-coital times of the pregnant females of the control group (1 to 4 test days).
- 75 and 250 mg/kg bw/day: no test item-related differences were noted for the length of the gestation period between the rats of the control group and the treated rats. The mean gestation length of the animals of the 250 mg/kg bw/day dose group was slightly above the laboratory background range. However, this slight elongation of the gestation length, which was without statistical significance was considered to be spontaneous.
- 75 and 250 mg/kg bw/day: no test item-related differences were noted for the mean number of implantation sites between the control group and dose groups. Furthermore, correlating, the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the dose groups.

T4 HORMONE DETERMINATION.
Males:
- 75, 250 and 750/500 mg/kg bw/day: no toxicologically relevant changes were noted for the T4 levels of all treatment groups.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
Critical effects observed:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Post cull period:
- 75, 250 and 750 mg/kg bw/day: a test item-related decreased viability index was noted for all test item treated groups. In detail, dead pups were noted for 5 of 9 dams of the 75 mg/kg bw/day dose group, 9 of 9 dams of the 250 mg/kg bw/day dose group and for the two remaining dams of the 750 mg/kg bw/day dose group with live pups during the post cull period. In contrast, in the control group dead pups were only noted for 2 of 8 dams.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
MORTALITY / VIABILITY:
- 75 and 250 mg/kg bw/day: no test item-related differences were noted for the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the dose groups.
Pre-culled period:
- 75 and 250 mg/kg bw/day: no difference between the control group and the two treatment groups was noted for the viability index between lactation days 1 and 4.
- 750 mg/kg bw/day: most of the live born pups died between lactation days 0/1 and 4 (46 prematurely deceased pups of 62 live born pups). The high mortality of the pups was due to bad health conditions of the dams (demonstrated by the premature deaths of altogether 6 dams at the begin or during the lactation period) and bad health conditions of the pups (demonstrated by a reduced body weight on lactation day 1). The overview of the litter fate is as follows:
- 2 of 10 pregnant females did not deliver live pups (one female only delivered stillbirths and another female died during littering).
- 8 of 10 pregnant females delivered live pups.
- 5 of 8 females with live pups showed a total litter loss during the pre-cull period. Three of these 5 females died a few days after the death of all their pups.
- 1 female was found dead (on lactation day 2) and the pups were prematurely sacrificed.
- 1 female lost all pups during the pre- and the post cull period.
- 1 female lost no pups during the pre-cull period but most of the pups during the post-cull period. However, the female was the only female with surviving pups at the end of the lactation period.

BODY WEIGHTS AND WEIGHT CHANGES:
1) Pups:
- 75 and 250 mg/kg bw/day: no test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the dams of the 75 mg/kg bw/day and the 250 mg/kg bw/day dose group on lactation days 1, 4 and 13. The reduction in pup body weight in comparison to the control group that was noted at the 250 mg/kg bw/day dose level on lactation day 4 (13.5 % below the value of the control group; males and females combined) was considered to be spontaneous as it was only slight and statistically not significant.
- 750 mg/kg bw/day: a statistically significantly and markedly reduced pup body weight was noted on lactation day 1 (p ≤ 0.01). The difference in pup body weight between the pups of treatment group and the pups of the control group increased during the further course of the lactation period. However, only a few pups of the dose group were still available during the further course of the lactation period (16 available pups on lactation day 4 and 3 available pups on lactation day 13).
- 75, 250 and 750 mg/kg bw/day: no runt was noted in the control group and in the treatment groups.

2) Litter:
- 75 and 250 mg/kg bw/day: statistically significantly (p ≤ 0.05 / 0.01) reduced litter weights were noted on lactation day 13 (male and female pups combined), due to the high number of prematurely deceased pups between lactation day 4 and 13.
- 750 mg/kg bw/day: the litter weight was statistically significantly (p ≤ 0.05 or p ≤ 0.01) reduced on lactation days 1, 4 and 13 (male and female pups combined) due to the reduced number of live born pups and the reduced pup body weights. However, at the dose level only 2 litters were still available on lactation day 4 and only 1 on lactation day 13.

GROSS PATHOLOGICAL FINDINGS:
- 75, 250 and 750 mg/kg bw/day: no gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with the test item after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period .

ORGAN WEIGHT FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIO.
- 75 and 250 mg/kg bw/day: the weight of the thyroid glands of the pups revealed no test item-related differences between the control group and the pups of the treatment groups.
- 750 mg/kg bw/day: only the values from the pups of one dam were available. They were 19.0 % (left thyroids of the pups) or 42.5 % (right thyroids of the pups) below the values of the control group. This maybe due to the reduced body weight of both pups. However, due to the low number of available pups at the high dose level, an assessment about an effect on the thyroid weight is not possible.

HISTOPATHOLOGICAL FINDINGS
- 75 and 250 mg/kg bw/day: no test item-related changes were noted for the thyroid glands from the pups of the control group and the pups from the treatment groups.
- 750 mg/kg bw/day: thyroid glands from the 2 pups of one dam revealed no test item-related changes.

MALE TO FEMALE RATIO OF THE PUPS:
- 75, 250 and 750 mg/kg bw: no test item-related influence on the male to female ratio was noted for all treatment groups. However, male to female ratios that markedly differed from the expected value (around 1.0) were noted at the 75 mg/kg bw/day and the 750 mg/kg bw/day dose group with opposite directions (increase and decrease value, respectively). However, the values of the male to female ratios that were noted for the 75 mg/kg bw/day and the 750 mg/kg bw/day dose group were within (low dose group) or only slightly below (high dose group) the range of laboratory background data. Furthermore, no consistent trend was noted. Whereas at the 75 mg/kg bw/day dose level more male pups as female pups were noted, the opposite result was noted at the 750 mg/kg bw/day dose level. Hence, the differences from the expected ratio were considered to be spontaneous.

NUMBER OF LIVE PUPS:
- 75 and 250 mg/kg bw/day: statistically significantly (p ≤ 0.05 / 0.01) reduced numbers of live pups (male and female pups combined) in comparison to the control group were noted on lactation day 13 at the 75 mg/kg bw/day and the 250 mg/kg bw/day dose level. This was due to the high number of prematurely deceased pups between lactation day 4 and 13.
- 750 mg/kg bw/day: statistically significantly reduced numbers of live pups (male and female pups combined) were already noted on lactation day 1 (p ≤ 0.01), due to the reduced number of live born pups.

ANO-GENITAL DISTANCE:
- 75, 250, and 750 mg/kg bw/day: no test item-related influence was noted on the absolute and the relative ano-genital distance of the male and the female pups of all treatment groups.
However, whereas no statistically significant differences between the control group and the treatment groups were noted for the absolute ano-genital distance, statistically significantly increased values were noted for the relative ano-genital distance of the male pups.
In detail, the relative ano-genital distance of the male pups was 12.0 % above the value of the control group (p ≤ 0.05) at the 250 mg/kg bw/day dose level and 27.2 % above the value of the control group (p ≤ 0.01) at the 750 mg/kg bw/day dose level.
Similarly increased values were noted for the relative ano-genital distance of the female pups of the 250 mg/kg bw/day and the 750 mg/kg bw/day dose group (10.9 % or 25.5 % above the value of the control group), but they failed statistical significance.
The differences that were noted at the 250 mg/kg bw/day and the750 mg/kg bw/day dose level for the relative ano-genital distance were due to a reduced pup body weight that was noted at the 250 mg/kg bw/day (considered as spontaneously) and the 750 mg/kg bw/day dose level (considered as test item-related). Hence, the increased ano-genital distance at the 250 mg/kg bw/day and the 750 mg/kg bw/day dose level was a secondary effect of the reduced pup body weight and not a test item-related effect on genital development. Furthermore, all absolute values of the ano-genital distance from the animals of the 250 mg/kg bw/day and the 750 mg/kg bw/day dose group were inside the range of the laboratory background data.

EXAMINATION OF THE MALE PUPS FOR NIPPLES:
- 75, 250 and 750 mg/kg bw/day: no test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups.

T4 HORMONE DETERMINATION:
- 75 and 250 mg/kg bw/day: no test item-related changes were noted for the T4 levels of the male and female pups.
- 750 mg/kg bw/day: T4 levels of one examined male and one examined female pup from one dam were available, which were markedly below the control group. However, an assessment about an effect on the T4 level is not possible with such a small number of examined pups.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Critical effects observed:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Conclusions:
Parental females
Fertility of male and female animals as unaffected during the course of this study. An elongated gestation length and a reduced gestation index was noted for the female animals at 750 mg test item/kg bw/day, which is attributed to the overt toxicity with mortality (6 out of 10 animals died) and should therefore not be considered as primariy reprotoxic effect.
Cesium fluoroaluminate is not considered to be a primary reprotoxic substance, since adverse effects on the fertility were only seen at signs of excessive toxicity, involving death of 6 of 10 female and 2 of 10 animals. Consequently, the criterial for classification and labelling as reprotoxic (impairment of fertility) are not met.

Pups
No adverse effect was noted on the prenatal development of the pups at these dose levels. At 75 and 250 mg test item/kg bw/day a reduced viability index as observed during the post-cull period. At the maternal lethal dose of 750 mg test item/kg b.w./day an adverse effect on the prenatal development was noted in the form of a reduced number of implantation sites, an increased number of resorptions and stillbirths and a reduced viability index during the pre- and post-cull period.
Cesium fluoroaluminate caused a dose-dependent decrease of the viability index during the post-cull period, starting at the lowest dose administered. Due to the unclear etiology (either being a direct developmental toxicity or an increased sensitivity of the newborn being exposed via lactation), further studies are required before a definitive decision can be made with regard to classification and labelling.

Overall, a LOAEL of 75 mg/kg bw/day is derived for cesium fluoroaluminate based on a decreased pup viability index during the post-cull period.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Parental females

Fertility of male and female animals as unaffected during the course of this study. An elongated gestation length and a reduced gestation index was noted for the female animals at 750 mg test item/kg bw/day, which is attributed to the overt toxicity with mortality (6 out of 10 animals died) and should therefore not be considered as primary reprotoxic effect.

Cesium fluoroaluminate is not considered to be a primary reprotoxic substance, since adverse effects on the fertility were only seen at signs of excessive toxicity, involving death of 6 of 10 female and 2 of 10 animals. Consequently, the criterial for classification and labelling as reprotoxic (impairment of fertility) are not met.

Pups

No adverse effect was noted on the prenatal development of the pups at these dose levels. At 75 and 250 mg test item/kg bw/day a reduced viability index as observed during the post-cull period. At the maternal lethal dose of 750 mg test item/kg b.w./day an adverse effect on the prenatal development was noted in the form of a reduced number of implantation sites, an increased number of resorptions and stillbirths and a reduced viability index during the pre- and post-cull period.

Cesium fluoroaluminate caused a dose-dependent decrease of the viability index during the post-cull period, starting at the lowest dose administered. Due to the unclear etiology (either being a direct developmental toxicity or an increased sensitivity of the newborn being exposed via lactation), further studies are required before a definitive decision can be made with regard to classification and labelling.

Overall, a LOAEL of 75 mg/kg bw/day is derived for cesium fluoroaluminate based on a decreased pup viability index during the post-cull period.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Cesium fluoroaluminate is not considered to be a primary reprotoxic substance, since adverse effects on the fertility were only seen at signs of excessive toxicity, involving death of 6 of 10 female and 2 of 10 animals. The criteria for classification and labelling in accordance with regulation EC 1272/2008 are not met, hence no classification as reprotoxic (fertility impairment) required.

Cesium fluoroaluminate caused a dose-dependent decrease of the viability index during the post-cull period, starting at the lowest dose administered. Due to the unclear etiology (either being a direct developmental toxicity or an increased sensitivity of the newborn being exposed via lactation), further studies are required before a definitive decision can be made with regard to classification and labelling for the endpoint reproductive toxicity (developmental toxicity).