Registration Dossier

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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Jul 2016 to 10 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
sub-acute
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Jul 2016 to 10 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same 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:
adopted 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Envigo Research Limited, Shardlow Business Park, Shardlow Derbyshire, DE72 2GD UK
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: approximately 11 weeks (males) and approximately 14 weeks (females)
- Weight at study initiation: 276 to 357g (males) and 188 to 232g (females)
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. air changeEnvironmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet: pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) ab libitum
- Water: Mains drinking water was supplied ab libitum from polycarbonate bottles attached to the cage.
- Acclimation period: 19 days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01-07-2016 To: 24-09-2016
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Corn oil. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical S rvices. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark.

VEHICLE
- Concentration in vehicle: 12.5, 31.25 and 75 mg/mL for 50, 125 and 300 mg/kg bw/day dose levels respectively.
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken and analyzed for concentration of the test substance at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 6% of the nominal concentration and thus the required content limit of ± 10% with reference to nominal concentrations was met.

DETAILS ON ANALYTICAL METHOD
The concentration of test item in the final solution was quantified by GC using flame ionisation detection:
- GC system: Agilent Technologies 6890 incorporating autosampler and workstation
- Column: ZB-5 (30 m x 0.53 mm id x 5 µm film)
- Oven program: 150 °C for 0 min, with 10°C/ min to 230 °C then 50 °C/ min final temperature, 300 °C for 5 min
- Injection temperature: 250 °C
- Flame ionization detector temperature: 250 °C
- Injection volume: 1.0 µL
- Retention time: ~3.7 mins
Duration of treatment / exposure:
6 weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 male and 12 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen by the Sponsor and based on the available data from previous toxicity work including a 7 Day range-finding toxicity study in the rat (Envigo Study Number: MD65XP).
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS:
Behavioral assessment:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait (Tiptoe, High stepping, Spastic, Waddling, Dysmetric, Splayed, Scissor, Ataxic), Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation. This test was developed from the methods used by Irwin (1968) and Moser et al (1988).
Functional Performance Tests:
- Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
- Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
- Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7- 14 and 14-20. For females with live litters, food consumption was recorded on Days 1, 4, 7 and 14 post partum.

FOOD EFFICIENCY:
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

HAEMATOLOGY:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 43 for males and Day 13 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling. The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic). Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea Inorganic phosphorus (P), Glucose Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids Calcium. (Ca++).

THYRIOD HORMONE ANALYSIS
Where possible, blood samples were taken, allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormones from the following: All adult males at termination and all surviving adult females at termination.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Sacrifice and pathology:
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Surviving adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

GROSS PATHOLOGY:
- For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded
- All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
- The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group: Adrenals, Brain, Heart, Kidneys, Liver, Spleen, Thymus
- The following organs were weighed from all remaining animals: Cowpers Glands, Epididymides, Glans Penis, LABC (levator ani-bulbocavernous muscle), Ovaries, Pituitary (post-fixation), Prostate, Seminal Vesicles (with Coagulating Gland), Testes, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix)

HISTOPATHOLOGY:
- Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Esophagus, Eyes (fixed in Davidson’s fluid), Heart, Ileum (including peyer’s patches), Jejunum, Kidneys, Liver, Lungs (with bronchi) (were inflated to approximately normal respiratory volume with buffered 10% formalin before immersion in fixative), Lymph nodes (mandibular and mesenteric), Muscle (skeletal), Pancreas, Rectum, Salivary glands (submaxillary), Sciatic nerve, Skin, Spinal cord (cervical, mid thoracic and lumbar), Spleen, Stomach, Trachea, Thymus, Urinary bladder.
- Samples of the following tissues were preserved for all remaining animals: Coagulating gland, Cowpers glands, Right Epididymides (preserved in Modified Davidsons fluid), Glans penis, Gross lesions, LABC (levator ani-bulbocavernous) muscle, Mammary gland, Ovaries, Pituitary, Prostate, Seminal vesicles, Right Testes (preserved in Modified Davidsons fluid), Thyroid/Parathyroid, Uterus & Cervix, Vagina
The tissues from five selected control and 300 mg/kg bw/day dose group animals and any animals dying during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues preserved for all animals from the remaining control and 300 mg/kg bw/day animals were also processed. In addition, sections of testes from all control and 300 mg/kg bw/day males were also stained with Periodic Acid- Schiff (PAS) stain and examined. Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post Implantation Loss, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Hematology, Blood Chemistry, Absolute Organ Weight s, Body Weight-Relative Organ Weights, Thyroid Hormone Analysis.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module. See 'Any other information on materials and methods incl. tables' for more details.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 300 mg/kg bw/day showed increased salivation from Day 7 to Day 44 (males) and Day 51 (females). One male treated with 125 mg/kg bw/day also showed increased salivation on Days 14 and 16 only. No such effects were detected in females treated with 125 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day. The control female that was killed in extremis on Day 39 showed pilo-erection, hunched posture and lethargy prior to termination.
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related deaths. One control female was killed in extremis on Day 39 due to adverse clinical signs. Microscopically this female had general signs of malaise with thymic atrophy and lymphoid depletion. There was significant inflammatory change in the uterus and this was considered to be the major factor contributing to death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males treated with 300 mg/kg bw/day showed a slight reduction (10%) in overall body weight gain. No statistically significant differences in body weight gain were evident in these males throughout the treatment period and no adverse clinical signs were detected. The slight reduction in overall body weight gain was therefore considered not to represent an adverse effect of treatment. No adverse effects were detected in females treated with 300 mg/kg bw/day or in animals of either sex treated with 125 or 50 mg/kg bw/day. Females treated with 50 mg/kg bw/day showed a statistically significant reduction (p<0.01) in cumulative body weight gain between Days 0 and 20 of gestation. In the absence of a similar effect in females treated with 125 or 300 mg/kg bw/day, the intergroup difference, in isolation, was considered to be of no toxicological importance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no adverse effect of treatment on food consumption in animals of either sex.
Females treated with 300 and 125 mg/kg bw/day showed a statistically significant reduction (p<0.05) in food consumption during the first week of gestation. Full recovery was evident thereafter and food consumption values during maturation were also comparable to controls. The intergroup differences were therefore considered to be of no toxicological significance. A statistically significant increase (p<0.01) in food consumption was also evident in females treated with 125 mg/kg bw/day between Days 1 and 4 of lactation however an increase in food consumption is generally considered not to be an adverse effect.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on food conversion efficiency (where calculated) in animals of either sex.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any intergroup differences in water intake for animals of either sex given the test item in relation to controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the haematology parameters examined. Males treated with 300 mg/kg bw/day showed statistically significant increases in mean corpuscular volume (p<0.05) and reticulocyte count (p<0.01). Males from all treatment groups also showed a statistically significant reduction in mean corpuscular hemoglobin concentration (p<0.05). Females from all treatment groups showed a statistically significant increase in reticulocyte count (p<0.05). All of the individual values were within background control ranges and in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined. Males treated with 300 mg/kg bw/day showed statistically significant increases in aspartate aminotransferase (p<0.05), alanine aminotransferase (p<0.01), creatinine (p<0.01), albumin/ globulin ratio (p<0.05) and a statistically significant reduction in bilirubin (p<0.05). The effect on albumin/globulin ratio and bilirubin extended to males treated with 125 and 50 mg/kg bw/day. Males treated with 125 mg/kg bw/day also showed a statistically significant reduction (p<0.05) in creatinine. The majority of the individual values were within background control ranges and in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in the behavioral parameters considered to be related to treatment at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the organ weights measured. Females from all treatment groups showed a statistically significant increase (p<0.05-0.01) in liver weight: both absolute and relative to terminal body weight. Females treated with 125 mg/kg bw/day also showed a statistically significant reduction (p<0.01) in absolute and relative thymus weight. All of the individual values for thymus weights and all but one absolute and relative value for the liver weights were within historical control ranges. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological importance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings detected in surviving adult animals. The control female that was killed in extremis on Day 39 had yellow contents in the stomach, ileum and jejunum, red lungs, red/brown fluid in the uterus and five dead fetuses in the left uterine horn and eight dead fetuses in the right uterine horn. Microscopically this female had general signs of malaise with thymic atrophy and lymphoid depletion. There was significant inflammatory change in the uterus which was considered to be the major factor contributing to death. Incidental findings that were not associated with either a true dose related response or any histopathological correlates and were considered to be unrelated to treatment included small testes and epididymides (one control male), enlarged heart (one control female), flaccid testes (one male treated with 50 mg/kg bw/day), a small right seminal vesicle (one male treated with 125 mg/kg bw/day), increased renal pelvic space in the left kidney (one male treated with 125 mg/kg bw/day), enlarged, pale and fluid filled kidneys (one female treated with 125 mg/kg bw/day) and enlarged liver (one female treated with 300 mg/kg bw/day).
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no intergroup differences at any dose level considered to be related to treatment with the test item. During motor activity evaluations, females treated with 300 mg/kg bw/day showed a statistically significant reduction during the final 20% of activity monitoring time (p<0.05) when compared to controls. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup difference was considered not to be of toxicological significance. Sensory reactivity scores across all test item-treated dose groups were similar to controls.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected. There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle). No treatment-related effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
An evaluation of Thyroxine (T4) in adult males did not identify any treatment-related findings.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Conclusions:
Oral (gavage) administration of the test substance to Wistar Han™:RccHan™:WIST rats at dose levels of 0, 50, 125 and 300 mg/kg bw/day for 6 (males) to 8 (females) weeks did not result in any significant adverse toxicological effects. Therefore the no observed adverse effect level (NOAEL) is considered to be 300 mg/kg bw/day.
Executive summary:

For the test substance a Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test was performed in accordance with OECDTG422 and GLP. 12 male and 12 femaleWistar Han™:RccHan™:WIST rats were exposed daily to 0, 50, 125 and 300 mg/kg bw/day for 6 (male) or 8 (female) weeks. Prepared formulations were within ± 6% of the nominal concentration and thus the required content limit of ± 10% with reference to nominal concentrations was met. Animals were assessed for clinical signs, body and organ weight (changes), food consumption and efficiency, (neuro)behavioral signs and a panel of hematological, blood chemistry and pathological signs. In addition, thyroid hormone analysis was performed. Minor effects on food consumption in lactating females, a reduction in activity monitoring time and clinical signs were observed in treatment groups, however these effects were not considered to be adverse. Relative liver weights were increased up to 168% at the high dose. In absence of any other related biochemical or histopathological effects this increase in liver is to present a metabolic demand. No mortality or other significant adverse toxicological effects were observed in all dose groups. Therefore a NOAEL of 300 mg/kg bw/day (actual dose received) is derived based on the absence of toxicological relevant adverse effects.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Jul 2016 to 10 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Envigo Research Limited, Shardlow Business Park, Shardlow Derbyshire, DE72 2GD UK
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: 11 weeks (males) and 14 weeks (females)
- Weight at study initiation: 276 to 357g (males) and 188 to 232g (females)
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet: pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) ab libitum
- Water: Mains drinking water was supplied ab libitum from polycarbonate bottles attached to the cage.
- Acclimation period: 19 days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01-07-2016 To: 24-09-2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Corn oil. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical S rvices. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark.

VEHICLE
- Concentration in vehicle: 12.5, 31.25 and 75 mg/mL for 50, 125 and 300 mg/kg bw/day dose levels respectively.
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken and analyzed for concentration of the test substance at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 6% of the nominal concentration and thus the required content limit of ± 10% with reference to nominal concentrations was met.

DETAILS ON ANALYTICAL METHOD
The concentration of test item in the final solution was quantified by GC using flame ionization detection:
- GC system: Agilent Technologies 6890 incorporating autosampler and workstation
- Column: ZB-5 (30 m x 0.53 mm id x 5 µm film)
- Oven program: 150 °C for 0 min, with 10°C/ min to 230 °C then 50 °C/ min final temperature, 300 °C for 5 min
- Injection temperature: 250 °C
- Flame ionization detector temperature: 250 °C
- Injection volume: 1 µL
- Retention time: ~3.7 mins
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
Duration of treatment / exposure:
Up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen by the Sponsor and based on the available data from previous toxicity work including a 7 Day range-finding toxicity study in the rat (Envigo Study Number: MD65XP).
Maternal examinations:
CAGE SIDE OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS:
Behavioral assessment:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait (Tiptoe, High stepping, Spastic, Waddling, Dysmetric, Splayed, Scissor, Ataxic), Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation. This test was developed from the methods used by Irwin (1968) and Moser et al (1988).
Functional Performance Tests:
- Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
- Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
- Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7- 14 and 14-20. For females with live litters, food consumption was recorded on Days 1, 4, 7 and 14 post partum.

FOOD EFFICIENCY:
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

HAEMATOLOGY:
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 43 for males and Day 13 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling. The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic). Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea Inorganic phosphorus (P), Glucose Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids Calcium. (Ca++).

THYRIOD HORMONE ANALYSIS
Where possible, blood samples were taken, allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormones from the following: All adult males at termination and all surviving adult females at termination.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Surviving adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

GROSS PATHOLOGY:
- For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded
- All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
- The following organs were dissected free from fat and weighed before fixation from fiveselected males and five selected females from each dose group: Adrenals, Brain, Heart, Kidneys, Liver, Spleen, Thymus
- The following organs were weighed from all remaining animals: Cowpers Glands, Epididymides, Glans Penis, LABC (levator ani-bulbocavernous muscle), Ovaries, Pituitary (post-fixation), Prostate, Seminal Vesicles (with Coagulating Gland), Testes, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix)

HISTOPATHOLOGY:
- Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Esophagus, Eyes (fixed in Davidson’s fluid), Heart, Ileum (including peyer’s patches), Jejunum, Kidneys, Liver, Lungs (with bronchi) (were inflated to approximately normal respiratory volume with buffered 10% formalin before immersion in fixative), Lymph nodes (mandibular and mesenteric), Muscle (skeletal), Pancreas, Rectum, Salivary glands (submaxillary), Sciatic nerve, Skin, Spinal cord (cervical, mid thoracic and lumbar), Spleen, Stomach, Trachea, Thymus, Urinary bladder.
- Samples of the following tissues were preserved for all remaining animals: Coagulating gland, Cowpers glands, Right Epididymides (preserved in Modified Davidsons fluid), Glans penis, Gross lesions, LABC (levator ani-bulbocavernous) muscle, Mammary gland, Ovaries, Pituitary, Prostate, Seminal vesicles, Right Testes (preserved in Modified Davidsons fluid), Thyroid/Parathyroid, Uterus & Cervix, Vagina
The tissues from five selected control and 300 mg/kg bw/day dose group animals and any animals dying during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues preserved for all animals from the remaining control and 300 mg/kg bw/day animals were also processed. In addition, sections of testes from all control and 300 mg/kg bw/day males were also stained with Periodic Acid- Schiff (PAS) stain and examined. Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle.

Ovaries and uterine content:
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
PARAMETERS EXAMINED
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. For each litter the following was recorded:
- Number of offspring born
- Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
- Sex of offspring on Days 1, 4, 7 and 13 post partum
- Clinical condition of offspring from birth to Day 13 post partum
- Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)
Physical development:
- All live offspring were assessed for ano-genital distance on Day 1 post partum.
- Visible nipple count was performed for all male offspring on Day 13 post partum.

GROSS EXAMINATION OF DEAD PUPS:
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. For offspring used for blood sampling, a more limited gross necropsy, including internal confirmation of the sex of the offspring, was performed following blood sampling.

SACRIFICE
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. For offspring used for blood sampling, a more limited gross necropsy, including internal confirmation of the sex of the offspring, was performed following blood sampling.

GROSS NECROPSY
Identical to parental animals

HISTOPATHOLOGY / ORGAN WEIGTHS
No
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post Implantation Loss, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Hematology, Blood Chemistry, Absolute Organ Weight s, Body Weight-Relative Organ Weights, Thyroid Hormone Analysis.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module. See 'Any other information on materials and methods incl. tables' for more details.
Indices:
Post–implantation loss (%) = Number of implantation sites - Total number of offspring born/ Number of implantation sites x100

Live Birth Index (%) = Number of offspring alive on Day 1/ Number of offspring born x 100

Viability Index 1 (%) = Number of offspring alive on Day 4 / Number of offspring alive on Day 1 x 100

Viability Index 2 (%) = Number of offspring alive on Day 13/ Number of offspring alive on Day 4 x 100

Sex Ratio (% males)= Number of male offspring/ Total number of offspring x 100
Historical control data:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals treated with 300 mg/kg bw/day showed increased salivation from Day 7 to Day 51. The control female that was killed in extremis on Day 39 showed pilo-erection, hunched posture and lethargy prior to termination.
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related deaths. One control female was killed in extremis on Day 39 due to adverse clinical signs. Microscopically this female had general signs of malaise with thymic atrophy and lymphoid depletion. There was significant inflammatory change in the uterus and this was considered to be the major factor contributing to death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effects were detected in females treated with 300 mg/kg bw/day or in animals of either sex treated with 125 or 50 mg/kg bw/day. Females treated with 50 mg/kg bw/day showed a statistically significant reduction (p<0.01) in cumulative body weight gain between Days 0 and 20 of gestation. In the absence of a similar effect in females treated with 125 or 300 mg/kg bw/day, the intergroup difference, in isolation, was considered to be of no toxicological importance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no adverse effect of treatment on food consumption. Females treated with 300 and 125 mg/kg bw/day showed a statistically significant reduction (p<0.05) in food consumption during the first week of gestation. Full recovery was evident thereafter and food consumption values during maturation were also comparable to controls. The intergroup differences were therefore considered to be of no toxicological significance. A statistically significant increase (p<0.01) in food consumption was also evident in females treated with 125 mg/kg bw/day between Days 1 and 4 of lactation however an increase in food consumption is generally considered not to be an adverse effect.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on food conversion efficiency (where calculated).
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any intergroup differences in water intake.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
All of the individual values were within background control ranges and in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The majority of the individual values were within background control ranges and in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in the behavioral parameters considered to be related to treatment at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the organ weights measured. Females from all treatment groups showed a statistically significant increase (p<0.05-0.01) in liver weight: both absolute and relative to terminal body weight. Females treated with 125 mg/kg bw/day also showed a statistically significant reduction (p<0.01) in absolute and relative thymus weight. All of the individual values for thymus weights and all but one absolute and relative value for the liver weights were within historical control ranges. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological importance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings detected in surviving adult animals. The control female that was killed in extremis on Day 39 had yellow contents in the stomach, ileum and jejunum, red lungs, red/brown fluid in the uterus and five dead fetuses in the left uterine horn and eight dead fetuses in the right uterine horn. Microscopically this female hadgeneral signs of malaise with thymic atrophy and lymphoid depletion. There was significant inflammatory change in the uterus which was considered to be the major factor contributing to death. Incidental findings that were not associated with either a true dose related response or any histopathological correlates and were considered to be unrelated to treatment included small (one male treated with 50 mg/kg bw/day), a small right seminal vesicle (one male treated with 125 mg/kg bw/day), increased renal pelvic space in the left kidney (one male treated with 125 mg/kg bw/day), enlarged, pale and fluid filled kidneys (one female treated with 125 mg/kg bw/day) and enlarged liver (one female treated with 300 mg/kg bw/day).
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
During motor activity evaluations, females treated with 300 mg/kg bw/day showed a statistically significant reduction during the final 20% of activity monitoring time (p<0.05) when compared to controls. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup difference was considered not to be of toxicological significance. Sensory reactivity scores across all test item-treated dose groups were similar to controls.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.. No treatment-related effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no detrimental effect of treatment with the test item on the mean number of implantations, post-implantation loss at 50, 125 or 300 mg/kg bw/day.
Dead fetuses:
no effects observed
Description (incidence and severity):
No effects on live birth index was observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no differences in gestation lengths in animals receiving the test item when compared with controls.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): There were no differences in gestation lengths in animals receiving the test item when compared with controls.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no treatment-related effects in conception rates for test item-treated animals in relation to controls. All females were pregnant.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no indication of an effect of treatment with the test item on offspring body weights and body weight development up to Day 13 post partum. Litter weights for all treated dose groups were also comparable with controls.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There was no indication of an effect of treatment with the test item on offspring body weights and body weight development up to Day 13 post partum. Litter weights for all treated dose groups were also comparable with controls.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no detrimental effect of treatment with the test item on litter size
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no detrimental effect of treatment with the test item on sex ratio
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Of the litters born, litter size at birth from all treated dose groups was comparable with controls indicating the lack of any effect on offspring viability.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Of the litters born, litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum from all treated dose groups was comparable with controls indicating the lack of any effect on offspring viability.
External malformations:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 50, 125 or 300 mg/kg bw/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 50, 125 or 300 mg/kg bw/day.
Visceral malformations:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 50, 125 or 300 mg/kg bw/day.
Other effects:
no effects observed
Description (incidence and severity):
- An evaluation of Thyroxine (T4) in male/female offspring (Day 13 of age) did not identify any treatment-related findings.
- When compared with controls, evaluation of ano-genital distance on Day 1 post partum (male and female offspring) and visible nipple count on Day 13 post partum (male offspring) did not reveal any treatment-related intergroup differences.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Oral (gavage) administration of the test substance to Wistar Han™:RccHan™:WIST rats at dose levels of 0, 50, 125 and 300 mg/kg bw/day did not result in any significant adverse maternal toxicological effects and did not affect developmental parameters of the offspring. Therefore the no observed adverse effect level (NOAEL) is considered to be >=300 mg/kg bw/day.
Executive summary:

For the test substance a Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test was performed in accordance with OECDTG422 and GLP. 12 male and 12 femaleWistar Han™:RccHan™:WIST rats were exposed daily to 0, 50, 125 and 300 mg/kg bw/day for 6 (male) or 8 (female) weeks. Prepared formulations were within ± 6% of the nominal concentration and thus the required content limit of ± 10% with reference to nominal concentrations was met. Maternal animals were assessed for clinical signs, body and organ weight (changes), food consumption and efficiency, (neuro)behavioral signs and a panel of hematological, blood chemistry and pathological signs. In addition, thyroid hormone analysis was performed. Minor effects on food consumption in lactating females, a reduction in activity monitoring time, liver weight and clinical signs were observed in treatment groups, however these effects were not considered to be adverse. No mortality or other significant adverse toxicological effects were observed in all dose groups. In addition, no effects were observed on maternal developmental parameters (e.g. number of pregnant, number of implantations and post-implantation loss, effects on pregnancy duration and live birth index). For the offspring, no effects were observed for mortality and number of live offspring, bodyweight and weight changes, sex ratio, changes in litter size and weight and post-natal survival. In addition Macroscopic necropsy findings for offspring on the study were typical for the age observed. Therefore, a NOAEL of 300 mg/kg bw/day (actual dose received) is derived for maternal and developmental toxicity based on the absence of toxicological relevant adverse effects.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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:
adopted 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Envigo Research Limited, Shardlow Business Park, Shardlow Derbyshire, DE72 2GD UK
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
Reaction mass of Benzenepropanal, 4-​ethyl-​α,​α-​dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal
EC Number:
916-329-6
Molecular formula:
C13H18O
IUPAC Name:
Reaction mass of Benzenepropanal, 4-​ethyl-​α,​α-​dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal
Test material form:
liquid
Details on test material:
As described in 1.2 of dossier

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: 11 weeks (males) and 14 weeks (females)
- Weight at study initiation: 276 to 357g (males) and 188 to 232g (females)
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet: pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) ab libitum
- Water: Mains drinking water was supplied ab libitum from polycarbonate bottles attached to the cage.
- Acclimation period: 19 days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01-07-2016 To: 24-09-2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Corn oil. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical S rvices. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark.

VEHICLE
- Concentration in vehicle: 12.5, 31.25 and 75 mg/mL for 50, 125 and 300 mg/kg bw/day dose levels respectively.
- Amount of vehicle (if gavage): 4 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken and analyzed for concentration of the test substance at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 6% of the nominal concentration and thus the required content limit of ± 10% with reference to nominal concentrations was met.

DETAILS ON ANALYTICAL METHOD
The concentration of test item in the final solution was quantified by GC using flame ionisation detection:
- GC system: Agilent Technologies 6890 incorporating autosampler and workstation
- Column: ZB-5 (30 m x 0.53 mm id x 5 µm film)
- Oven program: 150 °C for 0 min, with 10°C/ min to 230 °C then 50 °C/ min final temperature, 300 °C for 5 min
- Injection temperature: 250 °C
- Flame ionization detector temperature: 250 °C
- Injection volume: 1 µL
- Retention time: ~3.7 mins
Duration of treatment / exposure:
6 weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
daily
Details on study schedule:
Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 male and 12 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen by the Sponsor and based on the available data from previous toxicity work including a 7 Day range-finding toxicity study in the rat (Envigo Study Number: MD65XP).
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS:
Behavioral assessment:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait (Tiptoe, High stepping, Spastic, Waddling, Dysmetric, Splayed, Scissor, Ataxic), Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation. This test was developed from the methods used by Irwin (1968) and Moser et al (1988). T
Functional Performance Tests:
- Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
- Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
- Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7- 14 and 14-20. For females with live litters, food consumption was recorded on Days 1, 4, 7 and 14 post partum.

FOOD EFFICIENCY:
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

HAEMATOLOGY:
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 43 for males and Day 13 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling. The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic). Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea Inorganic phosphorus (P), Glucose Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids Calcium. (Ca++).

THYRIOD HORMONE ANALYSIS
Where possible, blood samples were taken, allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormones from the following: All adult males at termination and all surviving adult females at termination.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.
Sperm parameters (parental animals):
- At necropsy, the left testis and epididymis were removed from all males, dissected from connective tissue and weighed separately.
- For the epididymis, the distal region was incised and a sample of the luminal fluid was collected and transferred to a buffer solution for analysis of sperm motility. The semen sample was assessed using an automated semen analyzer to determine the numbers of motile, progressively motile and non-motile sperm.
- For the testis, the tunica albuginea was removed and the testicular tissue was stored frozen at approximately -20°C. This was then thawed and homogenised in a suitable saline/detergent mixture and samples of the homogenate were examined to determine the number of homogenization resistant spermatids present.
- The cauda epididymis was separated from the body of the epididymis, weighed and then stored frozen at approximately -20ºC. This was then thawed and homogenised in an appropriate saline/detergent to determine the numbers of homogenization resistant spermatids.
- Morphological assessment was performed for all control and 300 mg/kg bw/day males on a sample of a minimum of 200 sperm, to determine the number with apparent structural anomalies.
Litter observations:
PARAMETERS EXAMINED
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. For each litter the following was recorded:
- Number of offspring born
- Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
- Sex of offspring on Days 1, 4, 7 and 13 post partum
- Clinical condition of offspring from birth to Day 13 post partum
- Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)
Physical development:
- All live offspring were assessed for ano-genital distance on Day 1 post partum.
- Visible nipple count was performed for all male offspring on Day 13 post partum.

GROSS EXAMINATION OF DEAD PUPS:
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. For offspring used for blood sampling, a more limited gross necropsy, including internal confirmation of the sex of the offspring, was performed following blood sampling.
Postmortem examinations (parental animals):
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Surviving adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

GROSS PATHOLOGY:
- For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded
- All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
- The following organs were dissected free from fat and weighed before fixation from fiveselected males and five selected females from each dose group: Adrenals, Brain, Heart, Kidneys, Liver, Spleen, Thymus
- The following organs were weighed from all remaining animals: Cowpers Glands, Epididymides, Glans Penis, LABC (levator ani-bulbocavernous muscle), Ovaries, Pituitary (post-fixation), Prostate, Seminal Vesicles (with Coagulating Gland), Testes, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix)

HISTOPATHOLOGY:
- Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Esophagus, Eyes (fixed in Davidson’s fluid), Heart, Ileum (including peyer’s patches), Jejunum, Kidneys, Liver, Lungs (with bronchi) (were inflated to approximately normal respiratory volume with buffered 10% formalin before immersion in fixative), Lymph nodes (mandibular and mesenteric), Muscle (skeletal), Pancreas, Rectum, Salivary glands (submaxillary), Sciatic nerve, Skin, Spinal cord (cervical, mid thoracic and lumbar), Spleen, Stomach, Trachea, Thymus, Urinary bladder.
- Samples of the following tissues were preserved for all remaining animals: Coagulating gland, Cowpers glands, Right Epididymides (preserved in Modified Davidsons fluid), Glans penis, Gross lesions, LABC (levator ani-bulbocavernous) muscle, Mammary gland, Ovaries, Pituitary, Prostate, Seminal vesicles, Right Testes (preserved in Modified Davidsons fluid), Thyroid/Parathyroid, Uterus & Cervix, Vagina
The tissues from five selected control and 300 mg/kg bw/day dose group animals and any animals dying during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues preserved for all animals from the remaining control and 300 mg/kg bw/day animals were also processed. In addition, sections of testes from all control and 300 mg/kg bw/day males were also stained with Periodic Acid- Schiff (PAS) stain and examined. Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
SACRIFICE
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. For offspring used for blood sampling, a more limited gross necropsy, including internal confirmation of the sex of the offspring, was performed following blood sampling.

GROSS NECROPSY
Identical to parental animals

HISTOPATHOLOGY / ORGAN WEIGTHS
No
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post Implantation Loss, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Hematology, Blood Chemistry, Absolute Organ Weight s, Body Weight-Relative Organ Weights, Thyroid Hormone Analysis.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module. See 'Any other information on materials and methods incl. tables' for more details.
Reproductive indices:
Pre-coital Interval = Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
Mating Index (%) = Number of animals mated / Number of animals paired x 100
Pregnancy Index (%) = Number of pregnant females / Number of animals mated x 100
Gestation Length = Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturition Index (%) = Number of females delivering live offspring / Number of pregnant females x 100
Offspring viability indices:
Post–implantation loss (%) = Number of implantation sites - Total number of offspring born / Number of implantation sites x100
Live Birth Index (%) = Number of offspring alive on Day 1 / Number of offspring born x 100
Viability Index 1 (%) = Number of offspring alive on Day 4 / Number of offspring alive on Day 1 x 100
Viability Index 2 (%) = Number of offspring alive on Day 13 / Number of offspring alive on Day 4 x 100
Sex Ratio (% males)= Number of male offspring / Total number of offspring x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 300 mg/kg bw/day showed increased salivation from Day 7 to Day 44 (males) and Day 51 (females). One male treated with 125 mg/kg bw/day also showed increased salivation on Days 14 and 16 only. No such effects were detected in females treated with 125 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day. The control female that was killed in extremis on Day 39 showed pilo-erection, hunched posture and lethargy prior to termination.
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related deaths. One control female was killed in extremis on Day 39 due to adverse clinical signs. Microscopically this female had general signs of malaise with thymic atrophy and lymphoid depletion. There was significant inflammatory change in the uterus and this was considered to be the major factor contributing to death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males treated with 300 mg/kg bw/day showed a slight reduction (10%) in overall body weight gain. No statistically significant differences in body weight gain were evident in these males throughout the treatment period and no adverse clinical signs were detected. The slight reduction in overall body weight gain was therefore considered not to represent an adverse effect of treatment. No adverse effects were detected in females treated with 300 mg/kg bw/day or in animals of either sex treated with 125 or 50 mg/kg bw/day. Females treated with 50 mg/kg bw/day showed a statistically significant reduction (p<0.01) in cumulative body weight gain between Days 0 and 20 of gestation. In the absence of a similar effect in females treated with 125 or 300 mg/kg bw/day, the intergroup difference, in isolation, was considered to be of no toxicological importance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no adverse effect of treatment on food consumption in animals of either sex.
Females treated with 300 and 125 mg/kg bw/day showed a statistically significant reduction (p<0.05) in food consumption during the first week of gestation. Full recovery was evident thereafter and food consumption values during maturation were also comparable to controls. The intergroup differences were therefore considered to be of no toxicological significance. A statistically significant increase (p<0.01) in food consumption was also evident in females treated with 125 mg/kg bw/day between Days 1 and 4 of lactation however an increase in food consumption is generally considered not to be an adverse effect.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on food conversion efficiency (where calculated) in animals of either sex.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any intergroup differences in water intake for animals of either sex given the test item in relation to controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematology parameters examined. Males treated with 300 mg/kg bw/day showed statistically significant increases in mean corpuscular volume (p<0.05) and reticulocyte count (p<0.01). Males from all treatment groups also showed a statistically significant reduction in mean corpuscular hemoglobin concentration (p<0.05). Females from all treatment groups showed a statistically significant increase in reticulocyte count (p<0.05). All of the individual values were within background control ranges and in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined. Males treated with 300 mg/kg bw/day showed statistically significant increases in aspartate aminotransferase (p<0.05), alanine aminotransferase (p<0.01), creatinine (p<0.01), albumin/ globulin ratio (p<0.05) and a statistically significant reduction in bilirubin (p<0.05). The effect on albumin/globulin ratio and bilirubin extended to males treated with 125 and 50 mg/kg bw/day. Males treated with 125 mg/kg bw/day also showed a statistically significant reduction (p<0.05) in creatinine. The majority of the individual values were within background control ranges and in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in the behavioral parameters considered to be related to treatment at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
During motor activity evaluations, females treated with 300 mg/kg bw/day showed a statistically significant reduction during the final 20% of activity monitoring time (p<0.05) when compared to controls. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup difference was considered not to be of toxicological significance. Sensory reactivity scores across all test item-treated dose groups were similar to controls.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected. There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle). No treatment-related effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
An evaluation of Thyroxine (T4) in adult males did not identify any treatment-related findings.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with all females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm evaluation at the end of the treatment period did not reveal any adverse effects on sperm concentration, motility, homogenization resistant spermatid counts or sperm morphology in treated males.
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on mating performance. All animals mated within the first four days after pairing. Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level. All females were pregnant.

Details on results (P0)

- Gestation lengths were between 22 and 24 days and the distribution of gestation lengths for treated females was essentially similar to control.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
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):
Clinical signs detected in pups from treated dose groups included small size, cold, no milk in stomach and found dead or missing. Such findings are often observed in this type of study and were considered not to be related to treatment with the test item.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Of the litters born, litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum from all treated dose groups was comparable with controls indicating the lack of any effect on offspring viability.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no indication of an effect of treatment with the test item on offspring body weights and body weight development up to Day 13 post partum. Litter weights for all treated dose groups were also comparable with controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 50, 125 or 300 mg/kg bw/day.
Other effects:
no effects observed
Description (incidence and severity):
- An evaluation of Thyroxine (T4) in male/female offspring (Day 13 of age) did not identify any treatment-related findings.
- When compared with controls, evaluation of ano-genital distance on Day 1 post partum (male and female offspring) and visible nipple count on Day 13 post partum (male offspring) did not reveal any treatment-related intergroup differences.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
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:
Oral (gavage) administration of the test substance to Wistar Han™:RccHan™:WIST rats at dose levels of 0, 50, 125 and 300 mg/kg bw/day for 6 (males) to 8 (females) weeks did not result in any significant adverse toxicological effects and did not affect reproductive performance nor fertility. In addition, for the offspring no significant toxicological effects were observed up to post-partum day 13. Therefore the no observed adverse effect level (NOAEL) is considered to be 300 mg/kg bw/day.
Executive summary:

For the test substance a Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test was performed in accordance with OECDTG 422 and GLP. 12 male and 12 femaleWistar Han™:RccHan™:WIST rats were exposed daily to 0, 50, 125 and 300 mg/kg bw/day for 6 (male) or 8 (female) weeks. Prepared formulations were within ± 6% of the nominal concentration and thus the required content limit of ± 10% with reference to nominal concentrations was met. Animals were assessed for clinical signs, body and organ weight (changes), food consumption and efficiency, (neuro)behavioral signs and a panel of hematological, blood chemistry and pathological signs. In addition, thyroid hormone analysis was performed. Minor effects on food consumption in lactating females, a reduction in activity monitoring time, liver weight and clinical signs were observed in treatment groups, however these effects were not considered to be adverse. No mortality or other significant adverse toxicological effects were observed in all dose groups. In addition, no effects were observed on reproductive performance, sperm measures nor estrous cycle. For the offspring no mortality or changes in body weight were observed. In addition, no toxicological relevant clinical signs, gross pathological observations and alterations in thyroxin, ano-genital distance or male nipple count were observed. Therefore, a NOAEL of 300 mg/kg bw/day (actual dose received) is derived for parental toxicity and fertility based on the absence of toxicological relevant adverse effects.