Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-10 till 2016-06-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-10 till 2016-06-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
22 March 1996
GLP compliance:
yes (incl. QA statement)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Limit test:
no
Species:
rat
Strain:
Wistar
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: males 8 weeks, females 7 weeks (The age difference between males and females was deliberate, in order to avoid brother-sister mating).
- Weight at study initiation: mean males 305.53 – 306.66 g, mean females 188.74 – 191.97 g
- Housing: in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the premating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating (females found sperm-positive on the same date will be considered a ‘lot’) and by animal number (within each lot the mated females were housed in numerical order). After delivery, the cage containing the dam with a litter was transferred to another cage rack. The location of the dam with the litter in this cage rack was determined by the delivery date and the animal number.
- Diet: ad libitum, a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England)
- Water: ad libitum. domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC)
- Acclimation period: 17 days

DETAILS OF FOOD AND WATER QUALITY: Each batch of VRF1 (FG) diet is analysed by the supplier for nutrients and contaminants. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility. The supplier periodically (twice per year) analyses water samples taken at the premises for a limited number of physical, chemical and microbiological variables.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 April 2016 To: 09 June 2016 (males); 20, 24 and 27 June 2016 (females)
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
Experimental diets were prepared by mixing powdered VRF1 (FG) diet with the appropriate amounts of test substance. The diets were mixed in a mechanical blender. Fresh batches of the experimental diets were prepared on 3 May 2016 and on 2 June 2016 and stored in a freezer (<-18°C) in plastic bags in portions sufficient for three to four days. The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced twice per week with fresh portions from the freezer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From both batches of diets prepared in the study (3 May 2016 and on 2 June 2016) samples were taken and stored in a freezer (≤ -18°C). Analyses to determine the stability, homogeneity and content of the test substance in the test diets were conducted as indicated below:
- Concent: The content of the test substance at each dietary level was determined in the batches prepared on 3 May 2016 and on 2 June 2016.
- Homogeneity: The homogeneity (and content) of the test substance in the experimental diets was assessed in the first batch (3 May 2016), by analysing five samples (taken at different locations in the feed container) of each test diet in duplicate. One sample of the control diet was analysed.
- Stability: To demonstrate the stability of the test substance under experimental conditions, samples of the batch of test diets prepared on 3 May 2016 (low-dose diet, mid-dose diet and high-dose diet) were analyzed at t=0 and analyzed after storage in the animal room (in an open container) for 4 days, and after storage in a freezer (<-18 °C) for at least 5 weeks.
Duration of treatment / exposure:
Male animals were fed the experimental diets during a 2-week premating period, during mating and subsequently until sacrifice after at least 28 days of exposure. The female animals were fed the experimental diets during a 2-week premating period, during mating, gestation and lactation up to the day of sacrifice (at, or shortly after, day 4 of lactation).
Frequency of treatment:
Continuously
Dose / conc.:
1 000 mg/kg diet
Remarks:
equivalent to 59 and 72 mg/kg bw/day for males and females, respectively
Dose / conc.:
4 500 mg/kg diet
Remarks:
equivalent to 259 and 293 mg/kg bw/day for males and females, respectively
Dose / conc.:
13 000 mg/kg diet
Remarks:
equivalent to 714 and 790 mg/kg bw/day for males and females, respectively
No. of animals per sex per dose:
12
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale: the doses are based on the results of a dose-range finding study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS:
In addition to the above daily general clinical observations, detailed clinical examinations outside the home cage were performed on all rats of all groups prior to the first exposure and then once weekly throughout the study, up to the last week of the gestation period. In the last week of the study the detailed clinical examinations were part of the Functional Observational Battery tests (FOB) in the animals concerned.

BODY WEIGHT:
Body weights of male and female animals were recorded shortly before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on day 0 and 4 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION:
Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day. Food was refreshed twice weekly.

COMPOUND INTAKE:
The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentrations, the food consumption and the body weight of each study phase period for males and females separately.

HAEMATOLOGY:
Hematology was conducted in samples collected prior to the end of the premating period (on 24 May 2016, after 14 days of exposure) in 5 rats/sex/group. Blood was taken by orbita punction whilst under CO2/O2 anesthesia. For prothrombin time citrate was used as an anticoagulant. For all other parameters EDTA was used as anticoagulant. In each sample the following determinations were carried: hemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes),
prothrombin time, thrombocyte count, mean corpuscular volume (MCV; calculated), mean corpuscular hemoglobin (MCV; calculated), mean corpuscular hemoglobin concentration (MCHC; calculated)

CLINICAL CHEMISTRY:
Clinical chemistry was conducted in the same rats as indicated above (Haematology). Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. In each plasma sample the following determinations were carried out: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), potassium (K), chloride (Cl), inorganic phosphate (PO4)

NEUROBEHAVIOURAL EXAMINATION:
Functional Observational Battery (FOB) tests and spontaneous Motor Activity Assessment (MAA) were performed in 5 animals/sex/group shortly prior to sacrifice of the male and female rats. FOB tests and MAA were performed on the male animals with the lowest identification numbers in each cage, and on females with a litter. On the morning of testing (at least one hour prior to the start of the observations) the selected animals were placed individually in macrolon cages in a waiting area in the examination room. After testing the animals were returned to the experimental room.
- FOB: The FOB used in the laboratory is adapted from the WHO/IPCS Functional Observational Battery that was used in the Collaborative Study on Neurotoxicity Assessment sponsored by the International Programme on Chemical Safety of the World Health Organization. Details on the conduct of observations included in this battery and operational definitions of the different scores for each item are given in the FOB-manual entitled "Functional Observational Battery. Operational Definitions" (Lammers, 2000). The FOB is a series of non-invasive observational and interactive measures designed to assess the neurobehavioral and functional integrity of the rat. First, measurements were carried out in the cage. The rat’s posture, palpebral closure and the possible presence of clonic and tonic convulsions were recorded. Then the rat was removed from the cage and the ease of removal and handling were rated. Palpebral closure and any lacrimation or salivation were also rated, and the presence or absence of piloerection and vocalizations was recorded. In addition, other signs, such as changes in skin and fur, exophthalmus, crustiness around the eyes, bite marks on the tail or paws, missing toe nails or emaciation (shallow stomach, protruding spinal vertebrae) were recorded. The rat was then placed in an open arena (77 l x 55 w x 7 h cm) and observed for 3 minutes. Rears (both supported and unsupported) were counted. At the same time, gait characteristics were recorded and ranked, the ease with which the rat locomoted was ranked, and arousal was assessed and recorded. Further, the occurrence of clonic and/or tonic convulsions, stereotypies and bizarre behavior was recorded. At the end of the observation period, the number of faecal boluses and urine pools were recorded. Following this observation period, reflex testing was conducted. Reflex testing consisted of recording the rat's responses to the approach of a pencil, a touch of a pencil to the rump, a click stimulus, tail pinch, and the constriction of the pupil to light. Aerial righting was rated next. Forelimb and hindlimb gripstrength were measured. Three valid determinations (from a maximum of five attempts) were taken for each gripstrength measure. The rectal temperature was taken with the rat restrained by hand. Finally, the hindlimb feet were painted lightly and landing foot splay was measured.
- Motor activity: Motor activity was assessed following FOB testing. Changes in spontaneous motor activity were assessed using an automated quantitative microprocessorbased video image analysis system. Rats were placed individually in open roofed cages measuring 48.8 l x 44.7 w x 50 h cm on the insides and equipped with a video camera suspended above the test cage. The position of the rat was continuously monitored throughout the test session. Spontaneous motor activity was expressed as the total distance run in a 30 minute test period. In addition, habituation of activity was evaluated. To this end, each session was divided into 5 time blocks of 6 minutes each. Motor activity tests were recorded on DVD, in order to enable re-analysis of motor activity tests should that be necessary for technical reasons. However, re-analysis was not necessary. Therefore, recordings will be removed from the study dossier after submission of the final report. Squads of up to eight animals were monitored simultaneously. Dose groups were evenly distributed for motor activity test cage and for time as much as possible. Motor activity testing of a squad was conducted immediately after functional observations for that squad had finished.
Sacrifice and pathology:
GROSS PATHOLOGY:
All male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anesthesia at necropsy and then examined grossly for pathological changes. All males were sacrificed after the mating period on 9 June 2016 (after 30 days of treatment). The dams were sacrificed on day 4 of lactation or one day thereafter (e.g. those dose selected for FOB/motor activity testing). The dams and pups of the various lots were sacrificed between 20 – 27 June 2016.

Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde; except for the testes and the epididymides which were preserved in Bouin's fixative: testesm epididymides, prostate, ovaries (after counting the corpora lutea), uterus (after counting of the implantation sites), seminal vesicles (with coagulation glands).

In addition, the following organs of five parent animals/sex/group (males with the lowest identification numbers in each cage; females with a litter were selected) were preserved: adrenals, heart, kidneys, lungs, spleen, stomach, thymus, thyroid, urinary bladder, brain (including sections of cerebrum, cerebellum, medulla/pons), bone marrow (femur), liver, mesenterial and axillary lymph nodes, peripheral nerve (sciatic or tibial), small and large intestines (including Peyer’s patches), spinal cord (cervical, mid-thoracic, and lumbar), trachea.

HISTOPATHOLOGY:
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 μm, and stained with hematoxylin and eosin, except for sections of the testes which were stained with PAS hematoxylin. Microscopic examination was performed on the preserved organs of all animals of the control (group 1) and high-dose group (group 4). Since effects were observed on the microscopy of the kidneys of the male animals of the high-dose group, after consultation with the sponsor, the kidneys of the intermediate groups were also examined. Besides, immunohistochemistry for the α-microglobulin protein was performed on kidneys of 5 male animals per group. In brief, hereto the section of the kidneys were deparafinated and, after a series of washing and pre-treatment steps, incubated with a primary anti α2-microglobuline antibody (SSI, Denmark), washed again, and incubated with a peroxidase labelled second antibody (VWR, The Netherlands). Subsequently, the sections were contrastained, dehydrated and mounted. In addition, of rats of all dose groups organs showing gross lesions were microscopically examined

ORGAN WEIGHTS:
The testes and epididymis of all parent animals and the adrenals, heart, kidneys, spleen thymus, brain and liver of five animals/sex/group were weighed (paired organs together) as soon as possible after dissection to avoid drying.
Statistics:
See “Any other information on material and methods incl. tables”
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related clinical signs during the premating period, the mating period, post-mating period, the gestation period or the lactation period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- During the premating period, mean body weights of the male animals of the high-dose group were statistically significantly lower than control animals on day 7 and day 13 whereas no statistically significant effects were observed on the body weights of the female animals.
- During the pre-mating period, mean body weight changes of the male animals of the mid-dose group were statistically significantly lower than control animals from days 0 to 7 and from days 0 to 13. Mean body weight changes of the male animals of the high-dose group were statistically significantly lower than control animals from days 0 to 7, 7 to 13 and 0 to 13. No statistically significant effects on body weight changes of the female animals were observed.
- During the post-mating period, mean body weight and mean body weight changes of the male animals of the high-dose group were statistically significantly lower than control animals on day 0 and day 7 and from days 0 to 7, respectively. (day 0 post-mating is day 21 of the study).
- During the gestation period, mean body weights of the female animals of the mid-dose group were statistically significantly lower than control animals on day 14, mean body weights of the female animals of the high-dose groups were statistically significantly lower than control animals on days 7, 14 and 20.
- During the gestation period, mean body weight changes of the female animals of the mid-dose group were statistically significantly lower than control animals from days 7 to 14. In the high-dose group, mean body weight changes were statistically significantly lower than control animals from days 0-7, 7 to 14 and 0 to 20.
- During the lactation period, mean body weights of the female animals of the high-dose group were statistically significantly lower than control animals on day 0 and 4, whereas no statistically significant effects were observed on body weight changes.
- The statistically significant effects on body weights and body weight changes as observed in male and female animals of the high-dose group are considered as treatment related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
FOOD CONSUMPTION
- During the premating period, mean food consumption of the male animals of the mid-dose and high-dose groups and of the female animals of the high-dose group was statistically significantly lower than that of control animals from days 0 to 7 and from days 0 to 13.
- During the post-mating period, mean food consumption of the male animals of the high-dose group was statistically significantly lower than that of control animals from days 21 to 28.
- During the gestation period, mean food consumption of the female animals of the mid-dose group (from days 0 to 7, 7 to 14 and 0 to 20) and high-dose group (from days 0 to 7, 7 to 14, 14 to 20 and 0 to 20) were statistically significantly lower than that of control animals.
- During the lactation period, mean food consumption of the female animals of the high-dose group was statistically significantly lower than that of control animals.
- The decreased food consumption as observed in male and female animals of the high-dose group as compared to control animals was considered to be related to treatment.

COMPOUND INTAKE
- The test substance intake per kg body weight per day was calculated from the nominal dietary concentration, the food consumption and the body weight.
- During the premating period, the mean test substance intake of the male animals ranged from 60-65, 263-273 and 611-801 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
- During the premating period, the mean test substance intake of the female animals ranged from 70-73, 302-309 and 652-952 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
- During the post-mating period, the mean test substance intake of the male animals was 56, 250 and 732 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
- During the gestation period, the mean test substance intake of the female animals ranged from 69-80, 298-314 and 810-874 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
- During the lactation period, the mean test substance intake of the female animals was 110, 493 and 1231 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A slight, but statistically significantly, decrease in haemoglobin (Hb) concentration in the blood of male animals of the low-dose group was observed. Since no effects were observed in the mid- and high-dose groups, this finding was considered as fortuitously and not related to treatment. Overall, there were no treatment related changes in red blood cell clotting parameters. In the blood of the male animals of the mid-dose group, the % of lymphocytes was statistically significantly decreased whereas the % of neutrophils was statistically significantly increased. Since no effects were observed in the high-dose groups, this finding was considered as fortuitously and not related to treatment. Overall, there were no treatment related changes in total and differential white blood cell counts.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- In male animals, the concentration of creatinine and urea was statistically significantly increased in the mid- and high-dose groups. Plasma glucose levels were statistically significantly decreased in high-dose males.
- In female animals of the high-dose group, statistically significant increased concentrations of total protein, cholesterol and phospholipids were observed whereas the ratio albumin/globulin was statistically significantly decreased.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- FOB: No treatment-related effects were observed from detailed clinical observations or from functional observations in any of the dose groups during the study period. In some females from the control, low-, mid-, and high-dose groups slightly tiptoe walking was observed in different weeks of the study. Tiptoe walking was not considered to be related to treatment because it was not consistently observed in the concerned animals from first occurrence towards the end of the study, the severity did not increase over time and it was also observed in the control group. Tiptoe walking is considered to be related to estrus: it is part of the normal behavioral repertoire of the female rat, particularly during estrus. Some abnormalities (low or high handling reactivity, high activity in open field, nasal red discharge/encrustations, (small) dermal wound, encrustation near right eye, dermal encrustations and sparsely haired skin were observed in some animals from different groups in various weeks of the study. Based on the incidence and on the distribution among the dose groups, these findings were not ascribed to treatment. The observed anomalies right eye degeneration, mydriasis (right or both eyes), pupil reflex absent in right eye, right eyelid irritated and tilted head were not considered treatment-related. These anomalies were observed for the first time in week 3 and only in several rats that underwent orbita puncture (right eye) at the end of week 2 of the test period. Most likely, the orbita puncture caused mechanical damage in the right eye of the rats, which indirectly has resulted in the observed anomalies. During FOB testing, female rats from the low-dose group showed statistically significantly higher rearing. This finding is not considered treatment related because it is not observed in rats from the mid-dose and high-dose groups and because it is observed in one sex only. During neurobehavioral testing, it could not be prevented that the person executing the experiment was aware of the treatment of some of the animals.
- Motor Activity Assessment: No treatment-related effects were observed from motor activity assessment in any of the dose groups during the 30-minute test period. In males and females no statistically significant differences were observed between the dose groups and the control group in habituation of motor activity during the 30-minute test session in both females and males. Also no statistical significant differences were observed in the total distance moved.

Considering there were only a few remarkable observations using FOB-testing in this study, it is unlikely that the disclosure of the treatment of some of the animals had an effect on the outcome in this study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- In male animals of the high-dose group, terminal body weight was statistically significantly decreased and the relative weights of the epididymides and testes were statistically significantly increased whereas no effects on the absolute weights of these organs was observed.
- In female animals of the high-dose groups, terminal body weight and the absolute weight of the heart were statistically significantly decreased.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination revealed a dose - dependent increase of hyaline droplets in the lumen of the proximal tubular epithelium cells in the kidneys of the male animals. In the low-dose group, three out of five animals showed minimal to mild increase of hyaline droplets. In the mid-dose group, all five animals showed minimal to mild increase of hyaline droplets. In the high-dose group, all five animals showed mild increase of hyaline droplets. In addition, a dose-dependent increase of basophilic tubuli in the kidneys of the male animals was observed. In the low-dose group, two out of five animals showed mild basophilic tubuli. In the mid-dose group, four out of five animals showed minimal to mild basophilic tubuli. In the high dose group all five animals showed mild basophilic tubuli. Furthermore, a dose-dependent increase was observed of dilated tubuli, filled with eosinophilic debris (granular casts), in the kidneys of the male animals. No granular casts were observed in the control and the low-dose groups. In the mid-dose group, two out of five animals showed minimal to mild granular casts. In the high dose group, three out of five animals showed minimal to mild granular casts. The remaining histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age. The above histological observations are in agreement with the description of nephrotoxicity induced by the accumulation of a2-microglobulin in males, characterized by enlarged lysozomes, granular casts (as a result of the exfoliation of the proximal tubule epithelium) and tubular cell proliferation (basophilic tubules). To confirm the presence of a2-microglobulin, we have performed immunohistochemistry with a specific antibody against a2-microglobulin. For this technique, retrieval of the epitope is essential. The results show that the granular casts are positive for a2-microglobulin staining, indicative of a2-microglobulin accumulation in the exfoliated tubule cells. In addition, some hyaline droplets, but not all, are positive for a2-microglobulin staining.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
The relevance, of nephrotoxicity induced by the accumulation of α2-microglobulin, for humans is questionable.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg diet
Based on:
test mat.
Remarks:
equivalent to 59 mg/kg bw/day for males
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: the relevance for this finding in humans is questionable
Key result
Dose descriptor:
NOAEL
Effect level:
4 500 mg/kg diet
Based on:
test mat.
Remarks:
equivalent to 259 and 293 mg/kg bw/day for males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
Key result
Critical effects observed:
no
Conclusions:
Based on the effects on food consumption, body weights, clinical chemistry and organ weights as observed in the high-dose group, the NOAEL was placed at 4500 mg/kg diet (mid-dose; equivalent to a mean overall intake of 259 mg/kg bw/day in males and at least 293 mg/kg bw/day in females).
Executive summary:

In a GLP compliant study, performed according to OECD guideline 422, the possible effects of the test substance on general toxicity, reproductive performance and development of pups were examined in groups of 12 male and 12 female Wistar rats. The test substance was administered at constant concentrations in the diet at levels of 0 (control), 1000, 4500 and 13000 mg/kg diet during a premating period of 2 weeks and during mating, gestation until day 4 of lactation. Female animals and pups were sacrificed at or shortly after day 4 of lactation. Male animals were sacrificed after the mating period. The content, homogeneity and stability of the test substance in the carrier were confirmed by analysis. The dietary levels as applied in this study provided an overall mean test substance intake over the pre- and post-mating periods of 59 (range 56-65), 259 (range 250-273) and 714 (range 611-801) mg/kg bw/d in males of the low-, mid- and high-dose group, respectively. In females, the overall mean test substance intake during the premating period was 72 (range 70-73), 304 (range 302-309) and 790 (range 652-952) mg/kg bw/d in the low-, mid- and high-dose group, respectively. During the gestation period, the overall mean test substance intake was 72 (range 69-80), 293 (range 298-314) and 796 (range 810-874) mg/kg bw/d in the low-, mid- and high-dose group, respectively. During the lactation period, the overall mean test substance intake was 110, 493 and 1231 mg/kg bw/d in the low-, mid- and high-dose group, respectively. There was no treatment-related mortality. Daily clinical observations did not reveal any treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. The statistically significant effects on body weights, body weight changes and food consumption as observed in male and female animals of the high-dose group were considered to be related to treatment. Hematology and clinical chemistry was conducted in 5 rats/sex/group at the end of the premating period. No treatment-related effects were observed on hematology parameters. In the high-dose group, statistically significant effects on the concentrations of creatinine, urea and glucose in males and on the concentrations of total protein, cholesterol, phospholipids and on the ratio of albumin/globulin in female animals were observed. In high-dose males, the relative weight of the epididymides and testis were statistically significantly increased. In female animals, the absolute weight of the heart was statistically significantly decreased. Macroscopic examination did not reveal any treatment-related abnormalities. Microscopic examination revealed a dose-dependent increase of hyaline droplets in the lumen of the proximal tubular epithelium cells in the kidneys of the male animals. In the low-dose group, three out of five animals showed minimal to mild increase of hyaline droplets. In the mid-dose group, all five animals showed minimal to mild increase of hyaline droplets. In the high-dose group, all five animals showed mild increase of hyaline droplets. The results of the histopathological analysis are in agreement with nephrotoxicity, induced by the accumulation of α2-microglobulin in the male kidneys. As a result of the accumulation, cells of the tubuli died, exfoliated and formed aggregates of eosinophilic debris in dilated tubuli (granular casts). In agreement with this observation, the granular casts are positive for α2-microglobulin. Based on the statistically significant increased incidence of male animals of the mid- and high-dose groups showing α2-microglobulin accumulation in the kidneys, the NOAEL for parental toxicity would be placed at 1000 mg/kg diet (low-dose; equivalent to a mean overall intake of at 59 mg/kg bw/day in males and at least 72 mg/kg bw/day in females). However, the relevance for this finding for humans is questionable. Based on the effects on food consumption, body weights, clinical chemistry and organ weights as observed in the high-dose group, the NOAEL for parental toxicity was placed at 4500 mg/kg diet (mid-dose; equivalent to a mean overall intake of 259 mg/kg bw/day in males and at least 293 mg/kg bw/day in females).

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:
22 March 1996
GLP compliance:
yes (incl. QA statement)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol
EC Number:
944-553-4
Cas Number:
1632042-40-0
Molecular formula:
C11H20O
IUPAC Name:
1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol
Test material form:
solid
Details on test material:
- Substance name as cited in test report: FRET 11-0571
- Phystical state: white solid
- Storage conditions: 15 - 25 °C

Test animals

Species:
rat
Strain:
Wistar
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: males 8 weeks, females 7 weeks (The age difference between males and females was deliberate, in order to avoid brother-sister mating).
- Weight at study initiation: mean males 305.53 – 306.66 g, mean females 188.74 – 191.97 g
- Housing: in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the premating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating (females found sperm-positive on the same date will be considered a ‘lot’) and by animal number (within each lot the mated females were housed in numerical order). After delivery, the cage containing the dam with a litter was transferred to another cage rack. The location of the dam with the litter in this cage rack was determined by the delivery date and the animal number.
- Diet: ad libitum, a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England)
- Water: ad libitum. domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC)
- Acclimation period: 17 days

DETAILS OF FOOD AND WATER QUALITY: Each batch of VRF1 (FG) diet is analysed by the supplier for nutrients and contaminants. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility. The supplier periodically (twice per year) analyses water samples taken at the premises for a limited number of physical, chemical and microbiological variables.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 April 2016 To: 09 June 2016 (males); 20, 24 and 27 June 2016 (females)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
Experimental diets were prepared by mixing powdered VRF1 (FG) diet with the appropriate amounts of test substance. The diets were mixed in a mechanical blender. Fresh batches of the experimental diets were prepared on 3 May 2016 and on 2 June 2016 and stored in a freezer (<-18°C) in plastic bags in portions sufficient for three to four days. The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced twice per week with fresh portions from the freezer.
Details on mating procedure:
At the end of the premating period, each female was caged with one male from the same dose group. Animals were caged together until mating occurred. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day 0. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups. After the first week of the mating period all females, except for 3, were successfully mated with their assigned male. Female 21 of the control was not mated by male 22, female 57 of the mid-dose group was misjudged to be not mated by male 58 (but later, female 57 appeared to be pregnant) and female 83 of the high-dose group was not mated by male 84. Based on this number of mating pairs, the mating period was finalized after one week. Dams were allowed to raise their litter until sacrifice, four or five days after giving birth. Males were killed after at least 28 days of exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From both batches of diets prepared in the study (3 May 2016 and on 2 June 2016) samples were taken and stored in a freezer (≤ -18°C). Analyses to determine the stability, homogeneity and content of the test substance in the test diets were conducted as indicated below:
- Concent: The content of the test substance at each dietary level was determined in the batches prepared on 3 May 2016 and on 2 June 2016.
- Homogeneity: The homogeneity (and content) of the test substance in the experimental diets was assessed in the first batch (3 May 2016), by analysing five samples (taken at different locations in the feed container) of each test diet in duplicate. One sample of the control diet was analysed.
- Stability: To demonstrate the stability of the test substance under experimental conditions, samples of the batch of test diets prepared on 3 May 2016 (low-dose diet, mid-dose diet and high-dose diet) were analyzed at t=0 and analyzed after storage in the animal room (in an open container) for 4 days, and after storage in a freezer (<-18 °C) for at least 5 weeks.
Duration of treatment / exposure:
Male animals were fed the experimental diets during a 2-week premating period, during mating and subsequently until sacrifice after at least 28 days of exposure. The female animals were fed the experimental diets during a 2-week premating period, during mating, gestation and lactation up to the day of sacrifice (at, or shortly after, day 4 of lactation).
Frequency of treatment:
Continuously
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg diet
Remarks:
equivalent to 59 and 72 mg/kg bw/day for males and females, respectively
Dose / conc.:
4 500 mg/kg diet
Remarks:
equivalent to 259 and 293 mg/kg bw/day for males and females, respectively
Dose / conc.:
13 000 mg/kg diet
Remarks:
equivalent to 714 and 790 mg/kg bw/day for males and females, respectively
No. of animals per sex per dose:
12
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale: the doses are based on the results of a dose-range finding study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS:
In addition to the above daily general clinical observations, detailed clinical examinations outside the home cage were performed on all rats of all groups prior to the first exposure and then once weekly throughout the study, up to the last week of the gestation period. In the last week of the study the detailed clinical examinations were part of the Functional Observational Battery tests (FOB) in the animals concerned.

BODY WEIGHT:
Body weights of male and female animals were recorded shortly before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on day 0 and 4 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION:
Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day. Food was refreshed twice weekly.

COMPOUND INTAKE:
The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentrations, the food consumption and the body weight of each study phase period for males and females separately.

HAEMATOLOGY:
Hematology was conducted in samples collected prior to the end of the premating period (on
24 May 2016, after 14 days of exposure) in 5 rats/sex/group. Blood was taken by orbita punction whilst under CO2/O2 anesthesia. For prothrombin time citrate was used as an anticoagulant. For all other parameters EDTA was used as anticoagulant. In each sample the following determinations were carried: hemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes),
prothrombin time, thrombocyte count, mean corpuscular volume (MCV; calculated), mean corpuscular hemoglobin (MCV; calculated), mean corpuscular hemoglobin concentration (MCHC; calculated)

CLINICAL CHEMISTRY:
Clinical chemistry was conducted in the same rats as indicated above (Haematology). Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. In each plasma sample the following determinations were carried out: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), potassium (K), chloride (Cl), inorganic phosphate (PO4)

NEUROBEHAVIOURAL EXAMINATION:
Functional Observational Battery (FOB) tests and spontaneous Motor Activity Assessment (MAA) were performed in 5 animals/sex/group shortly prior to sacrifice of the male and female rats. FOB tests and MAA were performed on the male animals with the lowest identification numbers in each cage, and on females with a litter. On the morning of testing (at least one hour prior to the start of the observations) the selected animals were placed individually in macrolon cages in a waiting area in the examination room. After testing the animals were returned to the experimental room.
- FOB: The FOB used in the laboratory is adapted from the WHO/IPCS Functional Observational Battery that was used in the Collaborative Study on Neurotoxicity Assessment sponsored by the International Programme on Chemical Safety of the World Health Organization. Details on the conduct of observations included in this battery and operational definitions of the different scores for each item are given in the FOB-manual entitled "Functional Observational Battery. Operational Definitions" (Lammers, 2000). The FOB is a series of non-invasive observational and interactive measures designed to assess the neurobehavioral and functional integrity of the rat. First, measurements were carried out in the cage. The rat’s posture, palpebral closure and the possible presence of clonic and tonic convulsions were recorded. Then the rat was removed from the cage and the ease of removal and handling were rated. Palpebral closure and any lacrimation or salivation were also rated, and the presence or absence of piloerection and vocalizations was recorded. In addition, other signs, such as changes in skin and fur, exophthalmus, crustiness around the eyes, bite marks on the tail or paws, missing toe nails or emaciation (shallow stomach, protruding spinal vertebrae) were recorded. The rat was then placed in an open arena (77 l x 55 w x 7 h cm) and observed for 3 minutes. Rears (both supported and unsupported) were counted. At the same time, gait characteristics were recorded and ranked, the ease with which the rat locomoted was ranked, and arousal was assessed and recorded. Further, the occurrence of clonic and/or tonic convulsions, stereotypies and bizarre behavior was recorded. At the end of the observation period, the number of faecal boluses and urine pools were recorded. Following this observation period, reflex testing was conducted. Reflex testing consisted of recording the rat's responses to the approach of a pencil, a touch of a pencil to the rump, a click stimulus, tail pinch, and the constriction of the pupil to light. Aerial righting was rated next. Forelimb and hindlimb gripstrength were measured. Three valid determinations (from a maximum of five attempts) were taken for each gripstrength measure. The rectal temperature was taken with the rat restrained by hand. Finally, the hindlimb feet were painted lightly and landing foot splay was measured.
- Motor activity: Motor activity was assessed following FOB testing. Changes in spontaneous motor activity were assessed using an automated quantitative microprocessorbased video image analysis system. Rats were placed individually in open roofed cages measuring 48.8 l x 44.7 w x 50 h cm on the insides and equipped with a video camera suspended above the test cage. The position of the rat was continuously monitored throughout the test session. Spontaneous motor activity was expressed as the total distance run in a 30 minute test period. In addition, habituation of activity was evaluated. To this end, each session was divided into 5 time blocks of 6 minutes each. Motor activity tests were recorded on DVD, in order to enable re-analysis of motor activity tests should that be necessary for technical reasons. However, re-analysis was not necessary. Therefore, recordings will be removed from the study dossier after submission of the final report. Squads of up to eight animals were monitored simultaneously. Dose groups were evenly distributed for motor activity test cage and for time as much as possible. Motor activity testing of a squad was conducted immediately after functional observations for that squad had finished.
Oestrous cyclicity (parental animals):
At the day of necropsy, of each female a vaginal smear was taken and stored for possible determination of the estrus cycle. Since in this study there were no effects on uterus weight the smears were discarded.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight
Litter observations:
At necropsy of the surviving dams, pups were examined externally for gross abnormalities and killed by hypothermia whilst under CO2/O2 anesthesia.
Postmortem examinations (parental animals):
GROSS PATHOLOGY:
All male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anesthesia at necropsy and then examined grossly for pathological changes. All males were sacrificed after the mating period on 9 June 2016 (after 30 days of treatment). The dams were sacrificed on day 4 of lactation or one day thereafter (e.g. those dose selected for FOB/motor activity testing). The dams and pups of the various lots were sacrificed between 20 – 27 June 2016.

Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde; except for the testes and the epididymides which were preserved in Bouin's fixative: testesm epididymides, prostate, ovaries (after counting the corpora lutea), uterus (after counting of the implantation sites), seminal vesicles (with coagulation glands).

In addition, the following organs of five parent animals/sex/group (males with the lowest identification numbers in each cage; females with a litter were selected) were preserved: adrenals, heart, kidneys, lungs, spleen, stomach, thymus, thyroid, urinary bladder, brain (including sections of cerebrum, cerebellum, medulla/pons), bone marrow (femur), liver, mesenterial and axillary lymph nodes, peripheral nerve (sciatic or tibial), small and large intestines (including Peyer’s patches), spinal cord (cervical, mid-thoracic, and lumbar), trachea.

HISTOPATHOLOGY:
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 μm, and stained with hematoxylin and eosin, except for sections of the testes which were stained with PAS hematoxylin. Microscopic examination was performed on the preserved organs of all animals of the control (group 1) and high-dose group (group 4). Since effects were observed on the microscopy of the kidneys of the male animals of the high-dose group, after consultation with the sponsor, the kidneys of the intermediate groups were also examined. Besides, immunohistochemistry for the α-microglobulin protein was performed on kidneys of 5 male animals per group. In brief, hereto the section of the kidneys were deparafinated and, after a series of washing and pre-treatment steps, incubated with a primary anti α2-microglobuline antibody (SSI, Denmark), washed again, and incubated with a peroxidase labelled second antibody (VWR, The Netherlands). Subsequently, the sections were contrastained, dehydrated and mounted. In addition, of rats of all dose groups organs showing gross lesions were microscopically examined

ORGAN WEIGHTS:
The testes and epididymis of all parent animals and the adrenals, heart, kidneys, spleen thymus, brain and liver of five animals/sex/group were weighed (paired organs together) as soon as possible after dissection to avoid drying.
Statistics:
See “Any other information on material and methods incl. tables”
Reproductive indices:
- mating days until Day 0 pc = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- female mating index = (number of females inseminated/number of females placed with males) x 100
- male mating index = (number of males placed with females /number of females inseminated) x 100
- female fertility index = number of pregnant females*100/number of inseminated females
- male fertility index = number of males with pregnant females*100/number of males placed with females
- gestation index = (number of females with live pups / number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites) / number of corpora lutea] x 100
- prenatal loss = (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites
- perinatal loss = (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered.
Offspring viability indices:
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day 0 - 4 = (number of pup surviving 4 days/number of liveborn on day 0) x100
- sex ratio day 0 = [(number of live male or female pups on day 0 / number of live pups on day 0] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related clinical signs during the premating period, the mating period, post-mating period, the gestation period or the lactation period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- During the premating period, mean body weights of the male animals of the high-dose group were statistically significantly lower than control animals on day 7 and day 13 whereas no statistically significant effects were observed on the body weights of the female animals.
- During the pre-mating period, mean body weight changes of the male animals of the mid-dose group were statistically significantly lower than control animals from days 0 to 7 and from days 0 to 13. Mean body weight changes of the male animals of the high-dose group were statistically significantly lower than control animals from days 0 to 7, 7 to 13 and 0 to 13. No statistically significant effects on body weight changes of the female animals were observed.
- During the post-mating period, mean body weight and mean body weight changes of the male animals of the high-dose group were statistically significantly lower than control animals on day 0 and day 7 and from days 0 to 7, respectively. (day 0 post-mating is day 21 of the study).
- During the gestation period, mean body weights of the female animals of the mid-dose group were statistically significantly lower than control animals on day 14, mean body weights of the female animals of the high-dose groups were statistically significantly lower than control animals on days 7, 14 and 20.
- During the gestation period, mean body weight changes of the female animals of the mid-dose group were statistically significantly lower than control animals from days 7 to 14. In the high-dose group, mean body weight changes were statistically significantly lower than control animals from days 0-7, 7 to 14 and 0 to 20.
- During the lactation period, mean body weights of the female animals of the high-dose group were statistically significantly lower than control animals on day 0 and 4, whereas no statistically significant effects were observed on body weight changes.
- The statistically significant effects on body weights and body weight changes as observed in male and female animals of the high-dose group are considered as treatment related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
FOOD CONSUMPTION
- During the premating period, mean food consumption of the male animals of the mid-dose and high-dose groups and of the female animals of the high-dose group was statistically significantly lower than that of control animals from days 0 to 7 and from days 0 to 13.
- During the post-mating period, mean food consumption of the male animals of the high-dose group was statistically significantly lower than that of control animals from days 21 to 28.
- During the gestation period, mean food consumption of the female animals of the mid-dose group (from days 0 to 7, 7 to 14 and 0 to 20) and high-dose group (from days 0 to 7, 7 to 14, 14 to 20 and 0 to 20) were statistically significantly lower than that of control animals.
- During the lactation period, mean food consumption of the female animals of the high-dose group was statistically significantly lower than that of control animals.
- The decreased food consumption as observed in male and female animals of the high-dose group as compared to control animals was considered to be related to treatment.

COMPOUND INTAKE
- The test substance intake per kg body weight per day was calculated from the nominal dietary concentration, the food consumption and the body weight.
- During the premating period, the mean test substance intake of the male animals ranged from 60-65, 263-273 and 611-801 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
- During the premating period, the mean test substance intake of the female animals ranged from 70-73, 302-309 and 652-952 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
- During the post-mating period, the mean test substance intake of the male animals was 56, 250 and 732 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
- During the gestation period, the mean test substance intake of the female animals ranged from 69-80, 298-314 and 810-874 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
- During the lactation period, the mean test substance intake of the female animals was 110, 493 and 1231 mg/kg body weight/day in the low-, mid- and high-dose groups, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A slight, but statistically significantly, decrease in haemoglobin (Hb) concentration in the blood of male animals of the low-dose group was observed. Since no effects were observed in the mid- and high-dose groups, this finding was considered as fortuitously and not related to treatment. Overall, there were no treatment related changes in red blood cell clotting parameters. In the blood of the male animals of the mid-dose group, the % of lymphocytes was statistically significantly decreased whereas the % of neutrophils was statistically significantly increased. Since no effects were observed in the high-dose groups, this finding was considered as fortuitously and not related to treatment. Overall, there were no treatment related changes in total and differential white blood cell counts.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- In male animals, the concentration of creatinine and urea was statistically significantly increased in the mid- and high-dose groups. Plasma glucose levels were statistically significantly decreased in high-dose males.
- In female animals of the high-dose group, statistically significant increased concentrations of total protein, cholesterol and phospholipids were observed whereas the ratio albumin/globulin was statistically significantly decreased.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- FOB: No treatment-related effects were observed from detailed clinical observations or from functional observations in any of the dose groups during the study period. In some females from the control, low-, mid-, and high-dose groups slightly tiptoe walking was observed in different weeks of the study. Tiptoe walking was not considered to be related to treatment because it was not consistently observed in the concerned animals from first occurrence towards the end of the study, the severity did not increase over time and it was also observed in the control group. Tiptoe walking is considered to be related to estrus: it is part of the normal behavioral repertoire of the female rat, particularly during estrus. Some abnormalities (low or high handling reactivity, high activity in open field, nasal red discharge/encrustations, (small) dermal wound, encrustation near right eye, dermal encrustations and sparsely haired skin were observed in some animals from different groups in various weeks of the study. Based on the incidence and on the distribution among the dose groups, these findings were not ascribed to treatment. The observed anomalies right eye degeneration, mydriasis (right or both eyes), pupil reflex absent in right eye, right eyelid irritated and tilted head were not considered treatment-related. These anomalies were observed for the first time in week 3 and only in several rats that underwent orbita puncture (right eye) at the end of week 2 of the test period. Most likely, the orbita puncture caused mechanical damage in the right eye of the rats, which indirectly has resulted in the observed anomalies. During FOB testing, female rats from the low-dose group showed statistically significantly higher rearing. This finding is not considered treatment related because it is not observed in rats from the mid-dose and high-dose groups and because it is observed in one sex only. During neurobehavioral testing, it could not be prevented that the person executing the experiment was aware of the treatment of some of the animals.
- Motor Activity Assessment: No treatment-related effects were observed from motor activity assessment in any of the dose groups during the 30-minute test period. In males and females no statistically significant differences were observed between the dose groups and the control group in habituation of motor activity during the 30-minute test session in both females and males. Also no statistical significant differences were observed in the total distance moved.

Considering there were only a few remarkable observations using FOB-testing in this study, it is unlikely that the disclosure of the treatment of some of the animals had an effect on the outcome in this study.
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):
Microscopic examination revealed a dose - dependent increase of hyaline droplets in the lumen of the proximal tubular epithelium cells in the kidneys of the male animals. In the low-dose group, three out of five animals showed minimal to mild increase of hyaline droplets. In the mid-dose group, all five animals showed minimal to mild increase of hyaline droplets. In the high-dose group, all five animals showed mild increase of hyaline droplets. In addition, a dose-dependent increase of basophilic tubuli in the kidneys of the male animals was observed. In the low-dose group, two out of five animals showed mild basophilic tubuli. In the mid-dose group, four out of five animals showed minimal to mild basophilic tubuli. In the high dose group all five animals showed mild basophilic tubuli. Furthermore, a dose-dependent increase was observed of dilated tubuli, filled with eosinophilic debris (granular casts), in the kidneys of the male animals. No granular casts were observed in the control and the low-dose groups. In the mid-dose group, two out of five animals showed minimal to mild granular casts. In the high dose group, three out of five animals showed minimal to mild granular casts. The remaining histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age. The above histological observations are in agreement with the description of nephrotoxicity induced by the accumulation of a2-microglobulin in males, characterized by enlarged lysozomes, granular casts (as a result of the exfoliation of the proximal tubule epithelium) and tubular cell proliferation (basophilic tubules). To confirm the presence of a2-microglobulin, we have performed immunohistochemistry with a specific antibody against a2-microglobulin. For this technique, retrieval of the epitope is essential. The results show that the granular casts are positive for a2-microglobulin staining, indicative of a2-microglobulin accumulation in the exfoliated tubule cells. In addition, some hyaline droplets, but not all, are positive for a2-microglobulin staining.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
At the day of necropsy, of each female a vaginal smear was taken and stored for possible determination of the estrus cycle. Since in this study there were no effects on uterus weight the smears were discarded.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
In male animals of the high-dose group, the relative weights of the epididymides and testes were statistically significantly increased whereas no effects on the absolute weights of these organs was observed.
Reproductive performance:
no effects observed
Description (incidence and severity):
Fertility was not affected by treatment. In each group 12 females were placed with males for mating. In the control group, one female was not mated whereas one female of the mid-dose was misjudged to be not-mated (daily vaginal smears did not reveal the presence of sperm cells but, nevertheless, this female appeared to be pregnant and delivered a litter). In contrary, in a vaginal smear of one female of the high-dose group sperm cells were observed but this female appeared to be not pregnant. Male and female mating and fertility indices were comparable among the groups. Reproductive performance was not affected by treatment. In each group the duration of gestation was comparable and the gestation index was 100% in all groups. No differences were observed on the mean number of corpora lutea and implantation sites between the groups and pre-implantation loss was not affected by treatment. There were no treatment related effects on prenatal loss and perinatal loss.

Effect levels (P0)

Key result
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related signs in pups during the lactation period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of pups delivered was comparable among the groups, no pups were stillborn. The number of pups that died or were missing were limited to 1 pup in the low-dose group and 1 pup in the mid-dose group. Consequently, the number of pups alive on day 4 was comparable among the groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related differences in mean pup weights between the test groups and the controls on day 0 or day 4 of lactation.
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:
no effects observed
Description (incidence and severity):
Sex ratio was comparable among the groups.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Two pups were missing that could not be macroscopically examined.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Generation:
F1
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In this study the test substance did not induce adverse effects to reproductive performance or fertility parameters, so the NOAEL was placed at ≥13000 mg/kg diet (the highest dose tested, equivalent to a mean overall intake of 714 mg/kg bw/day in males and at least 790 mg/kg bw/day in females).
Executive summary:

In a GLP compliant study, performed according to OECD guideline 422, the possible effects of the test substance on general toxicity, reproductive performance and development of pups were examined in groups of 12 male and 12 female Wistar rats. The test substance was administered at constant concentrations in the diet at levels of 0 (control), 1000, 4500 and 13000 mg/kg diet during a premating period of 2 weeks and during mating, gestation until day 4 of lactation. Female animals and pups were sacrificed at or shortly after day 4 of lactation. Male animals were sacrificed after the mating period. The content, homogeneity and stability of the test substance in the carrier were confirmed by analysis. The dietary levels as applied in this study provided an overall mean test substance intake over the pre- and post-mating periods of 59 (range 56-65), 259 (range 250-273) and 714 (range 611-801) mg/kg bw/d in males of the low-, mid- and high-dose group, respectively. In females, the overall mean test substance intake during the premating period was 72 (range 70-73), 304 (range 302-309) and 790 (range 652-952) mg/kg bw/d in the low-, mid- and high-dose group, respectively. During the gestation period, the overall mean test substance intake was 72 (range 69-80), 293 (range 298-314) and 796 (range 810-874) mg/kg bw/d in the low-, mid- and high-dose group, respectively. During the lactation period, the overall mean test substance intake was 110, 493 and 1231 mg/kg bw/d in the low-, mid- and high-dose group, respectively. There was no treatment-related mortality. Daily clinical observations did not reveal any treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. The statistically significant effects on body weights, body weight changes and food consumption as observed in male and female animals of the high-dose group were considered to be related to treatment. Hematology and clinical chemistry was conducted in 5 rats/sex/group at the end of the premating period. No treatment-related effects were observed on hematology parameters. In the high-dose group, statistically significant effects on the concentrations of creatinine, urea and glucose in males and on the concentrations of total protein, cholesterol, phospholipids and on the ratio of albumin/globulin in female animals were observed. In high-dose males, the relative weight of the epididymides and testis were statistically significantly increased. In female animals, the absolute weight of the heart was statistically significantly decreased. Macroscopic examination did not reveal any treatment-related abnormalities. Microscopic examination revealed a dose-dependent increase of hyaline droplets in the lumen of the proximal tubular epithelium cells in the kidneys of the male animals. In the low-dose group, three out of five animals showed minimal to mild increase of hyaline droplets. In the mid-dose group, all five animals showed minimal to mild increase of hyaline droplets. In the high-dose group, all five animals showed mild increase of hyaline droplets. The results of the histopathological analysis are in agreement with nephrotoxicity, induced by the accumulation of α2-microglobulin in the male kidneys. As a result of the accumulation, cells of the tubuli died, exfoliated and formed aggregates of eosinophilic debris in dilated tubuli (granular casts). In agreement with this observation, the granular casts are positive for α2-microglobulin. There were no effects of the test substance on fertility and reproductive performance, litter data or pup observations in any group. Based on the statistically significant increased incidence of male animals of the mid- and high-dose groups showing α2-microglobulin accumulation in the kidneys, the NOAEL for parental toxicity would be placed at 1000 mg/kg diet (low-dose; equivalent to a mean overall intake of at 59 mg/kg bw/day in males and at least 72 mg/kg bw/day in females). The relevance for this finding for humans is questionable. Based on the effects on food consumption, body weights, clinical chemistry and organ weights as observed in the high-dose group, the NOAEL for parental toxicity was placed at 4500 mg/kg diet (mid-dose; equivalent to a mean overall intake of 259 mg/kg bw/day in males and at least 293 mg/kg bw/day in females). Since there were no adverse effects on fertility parameters, reproductive performance and development the NOAEL for reproduction and developmental effects was placed at ≥ 13000 mg/kg diet (high-dose; equivalent to a mean overall intake of 714 mg/kg bw/day in males and at least 790 mg/kg bw/day in females).