Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 April 2015 to 29 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Dark yellow viscous liquid
- Analytical purity: 100%
- Expiry date: 02-Dec-2016
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
rat
Strain:
other: Sprague Dawley [Crl:CD(SD)]
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 66 males and 66 females received in good health from Charles River Laboratories, Inc., Raleigh, NC

- Weight at study initiation: males 309-400g, females 209-258 g. Female body weights ranged from 222-284g on gestation day 0

- Housing: Following receipt and until pairing, all F0 animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study.
During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding material. Following the breeding period, the males were individually housed in clean, solid-bottom cages until the scheduled necropsy. Following positive evidence of mating, the females were individually housed in plastic maternity cages with bedding material. The dams and their litters were housed in these cages until euthanasia on lactation day 4.
The 5 rats/sex in the control and high-dose groups that were assigned to the recovery phase were not paired for mating and remained housed in groups of 2-3 in clean solid-bottom cages until euthanasia

- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum throughout the acclimation period and during the study. Exception of all males and recovery phase females which were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

- Water (e.g. ad libitum): Reverse osmosis-purified water, delivered by an automatic watering system

- Acclimation period: 12 days

- Age at study initiation: approximately 10 weeks old for males and females at study day 0.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
-Vehicle was dispensed weekly for administration to the control group (Group 1) and for preparation of the test item formulations.
- Aliquots were prepared for daily dispensation to the control group and stored refrigerated, protected from light.
- Vehicle was mixed throughout the preparation sampling and dose administration procedures.
- Aliquots were heated in a water bath at 37 °C ± 5 °C on each dosing day prior to dispensation for dosing.

TEST ITEM
- Test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored refrigerated in the dark.
- Test item formulations were stirred continuously throughout the preparation sampling and dose administration procedures.
- Aliquots were heated in a water bath at 37 °C ± 5 °C on each dosing day prior to dispensation for dosing.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil was used as the substance formed stable homogenous solutions in this vehicle
- Concentration in vehicle: Dose-dependent (0 mg/mL, 20 mg/mL, 70 mg/mL, 100 mg/mL and 200 mg/mL).
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- Animals assigned to recovery were not evaluated for reproductive toxicity.
- M/F ratio per cage: the 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1 male:1 female basis within each dose group following 14 days of treatment for the males and females. Each female was cohabited with 1 male in a solid-bottom cage containing bedding material.
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: Presence of vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily.
- No replacement of first male by another male with proven fertility if unsuccessful mating. If no evidence of copulation detected after 14 days of pairing, females were placed in plastic maternity cages.
- After successful mating each pregnant female was caged (how): After evidence of mating (designated as gestation Day 0), the males were individually housed until euthanasia. Mated females were transferred to individual cages during gestation and lactation in solid-bottom cages with bedding material and remained in these cages with their litter until euthanasia on lactation Day 4/Postnatal Day4.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Test item formulations ranging from 10 to 220 mg/mL were previously assessed for homogeneity, resuspension homogeneity, and stability following 7 days of refrigerated storage.
- Samples for homogeneity and/or concentration determinations were collected from the top, middle and bottom strata of the first and last test item dosing formulations and from the middle stratum of the first and last control group dosing formulations.
- One set of samples from each collection was subjected to the appropriate analyses.
- All remaining samples were stored refrigerated as back-up.
- All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.
- The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%) and were homogeneous. The test item was not detected in the vehicle formulation that was administered to the control group (Group 1).
Duration of treatment / exposure:
Test duration:
- Control animals were treated in an identical manner with 5 mL/kg/day of Arachis oil.
- Males assigned to the recovery group were maintained for a further 15 days treatment-free period following termination of treatment for males (28 days).
- Females assigned to the recovery group were maintained for a further 15 days treatment-free period following termination of treatment for females (day of euthanasia of the first lactating female).
- The males selected for pairing were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
- The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-45 doses.
- The extra 5 rats/sex in the control and high-dose groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 28 doses for males and 39 doses for females, these animals remained on study for a 15-day recovery period.
Frequency of treatment:
The test and control substances were administered once daily, for 28 consecutive days (males) or 39-45 days (females), by gavage.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Details on study schedule:
- Animals were approximately 12 weeks old when paired on study day 13.
- Mated females were allowed to give birth and maintain their offspring until euthanasia on lactation Day 4/Postnatal Day4.
- During the period of parturition, females were observed twice daily for initiation and completion of parturition and for signs of dystocia
- On Day of parturition PND 0, pups were sexed and examined for gross malformations and the numbers of stillborn and live pups were recoreded.
- Individual gestation length was calculated using the date delivery started.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
350 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
15 animals of each sex (m/f) for groups 1 and 5 and 10 animals/sex/group for group 2 to 4. Based on the OECD guidelines 422 which recommends evaluation of at least 8 pregnant females per group.
In addition, 5 animals of each sex (m/f) were allocated to the control and high-dose group to assess recovery, persistence, or progression of any toxic effects following the cessation of dosing.
Control animals:
yes, concurrent vehicle
other: recovery control (concurrent vehicle)
Details on study design:
- Dose selection rationale: Results of a previous 14-day study in rats.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Regulatory requirement
- Post-exposure recovery period in satellite groups: 15 days, treatment free
Positive control:
Not used

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats observed twice daily, once in the morning and once in the afternoon, for appearance, behavior, moribundity, mortality and signs of overt toxicity. Individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT: Yes
- Time schedule for examinations: For males, recorded weekly throughout the study and prior to the scheduled euthanasia. For females, recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase).
Once evidence of mating observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 0 (when possible), 1 and 4.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing (for animals paired for breeding) or euthanasia (for animals assigned to the recovery period).
-Food consumption was measured on a per cage basis for the corresponding body weight intervals.
- Food consumption was normalized to the number of animals/cage and was reported in g/animal/day.
- Food intake was not recorded during the breeding period for animals selected for pairing. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4; food consumption was reported as g/animal/day and g/kg/day during gestation and lactation.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected from 5 animals/sex/group at the primary necropsy (study week 4 for males and lactation day 4 for females selected for pairing) and from 5 animals/sex in the control and high-dose groups at the recovery necropsy (following a 15-day recovery period; study day 42 for males and study day 53 for females).
- Blood for serum chemistry and hematology was collected from the retro-orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy.
- Animals fasted: Yes. Overnight prior to blood collection for all males and recovery phase females.
- Parameters examined: Total leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrombin time, activated partial thromboplastin time, reticulocyte count, mean platelet volume, differential leukocyte count (neutrophil, lymphocyte, monocyte, eosinophil, basophil, large unstained cell), red cell distribution width, hemoglobin distribution width, platelet estimate, red cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected from 5 animals/sex/group at the primary necropsy (study week 4 for males and lactation day 4 for females selected for pairing) and from 5 animals/sex in the control and high-dose groups at the recovery necropsy (following a 15-day recovery period; study day 42 for males and study day 53 for females).
- Animals fasted: Yes. Overnight prior to blood collection for all males and recovery phase females.
- Parameters examined: Albumin, total protein, globulin, albumin/globulin ratio, total bilirubin, urea nitrogen, creatinine, AP, ALAT, ASAT, gamma GT, glucose, total cholesterol, calcium, chloride, phosphorus, potassium, sodium, triglycerides, bile acids, appearance

OTHER:
NEUROBEHAVIOURAL EXAMINATION AND CAGE SIDE OBSERVATIONS: Yes
FOB Assessments
- Functional Observational battery were recorded for 5 animals/sex/group following approximately 28 days of dose administration (study week 4 for males selected for pairing, and on lactation day 4 for females).
- The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB.
- Battery of functions tested:
Home Cage Observations: Posture, convulsions/tremors, feces consistency, biting, palpebral (eyelid) closure)
Handling Observations: Ease of removal from cage, lacrimation/Chromodacryorrhea, piloerection, palpebral closure, eye prominence, red/crusty deposits, ease of handling animal in hand, salivation, fur appearance, respiratory rate/character, mucous membranes/eye/skin color, muscle tone
Open Filed Observations: mobility, rearing, convulsions/tremors, grooming, bizarre/stereotypic behavior, time to first step, grait, arousal, urination/defecation, grait score, backing
Sensory Observations: Approach response, startle response, pupil response, forelimb extension, air righting reflex, touch response, tail pinche response, eyeblink response, hindlimb extension, olfactory orientation
Neuromuscular Observations: Hindlimb extensor strenght, hindlimb foot splay, grip strenghts-hind and forelimb, rotarod performance
Physiological Observations: Catalepsy, Body weight, Body temperature

Motor Activity
- Motor activity was assessed for 5 animals/sex/group following approximately 28 days of dose administration (study week 4 for males selected for pairing, and on lactation day 4 for females).
- Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilizes a series of
infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity.
- The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB.
- Each animal was tested separately.
- Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes.
- These data were compiled as six, 10-minute subintervals for tabulation.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
LITTER VIABILITY AND DEATH
- Each litter was examined daily for survival and all deaths were recorded.
- Daily record of litter size was maintained.

CLINICAL OBSERVATIONS
- Litters were examined daily for survival and any adverse changes in appearance or behavior.
- Each pup received a clinical observation on PND 1 and 4

BODY WEIGHTS
- Pups were weighted on PND 1 and 4.

SEX DETERMINATION
- Pups were individually sexed on PND 0 and 4.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- A complete necropsy was conducted on all F0 parental animals at the scheduled termination. All F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period or following the 15-day recovery period. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded. Females with total litter loss were euthanized within 24 hours of litter loss. Females not selected for pairing were euthanized following the 15-day recovery period. Necropsy included examination of the external surface, all orifices, the cranialcavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.


HISTOPATHOLOGY: Yes
- At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted):
Adrenal glands, aorta, bone with marrow, brain, coagulating glands, eyes with optic nerve, gastroinstestinal tract (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum), heart, kidneys, liver, lymph node (axillary, mandibular, mesenteric), lungs, ovaries and oviduct, pancreas, peripheral nerve (sciatic), pituitary gland, prostate gland, salivary gland, seminal vesicles, skeletal muscle (rectus femoris), skin with mammary gland, spinal cord (cervical), spleen, testes with epididymides and vas deferens, thymus gland, thyroids, trachea, urinary bladder, uterus with cervix and vagina, all gross lesions
- Microscopic examination was performed on all tissues listed above from all animals in the control and 1000 mg/kg/day groups at the primary necropsy and on gross lesions from all animals in all groups at the scheduled necropsies.

The following organs were weighed from all F0 animals at the scheduled necropsies: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries with oviducts, spleen, testes, thymus gland, thyroids with parathyroids
Postmortem examinations (offspring):
SACRIFICE
- The surviving F1 offspring were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded on PND 4.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
ANALYSIS CONDUCTED BY WIL RESEARCH
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex.
Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, pre-coital intervals, gestation length, numbers of former implantation sites, corpora lutea, and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, and FOB data values were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. FOB parameters that yield scalar or descriptive data in the test item-treated groups were compared to the
control group using Fisher’s Exact test (Steel and Torrie, 1980). Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test item-treated groups to the control group.
Reproductive indices:
For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.
Mating, fertility, and copulation/conception indices were calculated as follows:

Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant) / Total No. of Males (Females) Used for Mating x 100

Male Fertility Index (%) = No.of Males Siring a Litter / Total No. of Males Used for Mating x 100

Male Copulation Index (%) = No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100

Female Fertility Index (%) = No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating x 100

Female Conception Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100
Confirmed Pregnancy)
Offspring viability indices:
Litter parameters were defined as follows:

Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter)* / No. of Litters Per Group x 100

Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)* / No. of Litters Per Group x 100

Where N = PND 0-1 and 1-4
* = Pups that were found dead due to drowning were excluded from pup viability calculations.

- Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia
- Litters euthanized prior to scheduled euthanasia due to reasons unrelated to test item administration were not considered to be a total litter loss on the data tables and were not included in the pup viability calculations.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
See results and table below.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
All males and females in the 100, 350, 500, and 1000 mg/kg/day groups survived to the scheduled necropsies. Test item-related clinical findings of clear and/or red material around the nose and mouth were noted for F0 males and females in all test item-treated groups approximately 1 hour following dose administration, generally throughout the dosing period. These findings generally did not persist to the daily examinations on the following days and in the absence of any other signs of toxicity for F0 animals, the aforementioned material findings were not considered to be adverse. Other clinical findings, including hair loss and yellow material on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related and therefore were not attributed to the test item. Clinical findings noted during the recovery period in the 1000 mg/kg/day group were limited to hair loss and
scabbing on the facial area for F0 females and scabbing on the dorsal trunk for F0 males.

REPRODUCTIVE PERFORMANCE
No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups.
The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.
-Gestation length and parturition: Mean gestation lengths in the 100, 350, 500, and 1000 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

BODY WEIGHT AND WEIGHT GAIN
- Males:
Mean body weights and body weight gains in the 100, 350, 500, and 1000 mg/kg/day group males were unaffected by test item administration throughout the study. None of the differences from the control group were statistically significant.
- Females during pre-mating:
Mean body weights and body weight gains in the 100, 350, 500, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating or entire dosing and recovery periods. None of the differences from the control group were statistically significant.
- Females during gestation:
Mean body weights and body weight gains in the 100, 350, 500, and 1000 mg/kg/day groups were unaffected by test item administration during gestation. Differences from the control group were slight and not statistically significant, with the following exceptions. In the 100 and 350 mg/kg/day groups, significantly (p<0.05 or p<0.01) higher mean body weight gains were noted during gestation days 17-20 and significantly (p<0.05 or p<0.01) higher mean body weights were generally noted for these groups throughout gestation. In addition, a significantly (p<0.05) higher mean body weight was noted for the 500 mg/kg/day group on gestation day 7. In the absence of a dose response, these higher mean body weights and body weight gains were not attributed to the test item.
- Females during lactation:
Mean body weights and body weight gains in the 100, 350, 500, and 1000 mg/kg/day groups were unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Males:
Mean food consumption, evaluated as g/animal/day, in the 100, 350, 500, and 1000 mg/kg/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed.
- Females during pre-mating:
Mean food consumption, evaluated as g/animal/day, in the 100, 350, 500, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating or entire dosing and recovery periods. None of the differences from the control group were statistically significant.
- Females during gestation:
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 350, 500, and 1000 mg/kg/day groups was unaffected by test item administration during gestation. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p<0.05 or p<0.01) lower mean g/kg/day values were noted for the 100, 350 (g/animal/day value was also significantly lower), and 1000 mg/kg/day groups compared to the control group during gestation days 7-11 and in the 350, 500, and 1000 mg/kg/day groups when the overall gestation period (days 0-20) was evaluated. Due to the lack of corresponding effects on body weights and body weight gains, these differences were not attributed to test item administration.
- Females during lactation:
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 350, 500, and 1000 mg/kg/day groups was unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

HAEMATOLOGY
There were no test item-related alterations in hematology and coagulation parameters noted at the primary necropsy. However, some statistically significant differences were observed when the control and test item-treated groups were compared. A lower mean corpuscular hemoglobin (MCH) value was noted in the 1000 mg/kg/day group females.
Measured values were similar to the control group and the difference was of minimal magnitude; therefore, the finding was attributed to biological variability. Statistically significant differences noted in the 100 and 500 mg/kg/day group males lacked a dose-response relationship, were of minimal magnitude change, and were not considered to be related to administration of the test item.
There were no test item-related alterations in hematology and coagulation parameters noted at the recovery necropsy. However, some statistically significant differences were observed when the control and test item-treated groups were compared. A higher red cell distribution width (RDW) value was noted in the 1000 mg/kg/day group males; measured parameters were similar to the control group and the difference was of minimal magnitude. Higher hemoglobin (HGB) and hemoglobin distribution width (HDW) values were noted in the 1000 mg/kg/day group females; other erythrocyte parameters were similar to the control group and the differences were of minimal magnitude. The differences were attributed to biological variability.

CLINICAL CHEMISTRY
Test item-related higher serum alanine aminotransferase (ALT) values were noted in the ≥350 mg/kg/day group males at the primary necropsy. Group mean values were higher than the WIL Research historical database range of study means, but there were no histologic correlates and the differences were of minimal magnitude; therefore, the findings were considered biologically irrelevant and non-adverse. There were no other test item-related alterations in serum chemistry parameters at the primary necropsy.
However, some statistically significant differences were observed when the control and test item-treated groups were compared. Lower serum ALT values were noted in the ≥500 mg/kg/day group females; the change was in a direction of no known toxicologic importance. Statistically significant differences noted in the 350 and 500 mg/kg/day group males lacked a dose-response relationship, were of minimal magnitude change, and were not considered to be related to administration of the test item.
There were no test item-related alterations in serum chemistry parameters at the recovery necropsy. However, some statistically significant differences were observed when the control and test item-treated groups were compared. A lower serum aspartate aminotransferase (AST) value were noted in the 1000 mg/kg/day group females; the change was in a direction of no known toxicologic importance. Lower serum phosphorus and higher triglyceride values were noted in the 1000 mg/kg/day group females; values at the PN were similar to the control group, the differences were of minimal magnitude, and were attributed to biological variability.


NEUROBEHAVIOUR
Functional Observational Battery (FOB)
- Home Cage Observations: Home cage parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Handling Observations: Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Open Field Observations: Open field parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Sensory Observations: Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Neuromusclar Observations: Neuromuscular parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Physiological Observations: Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Motor Activity: Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all concentrations when evaluated during study week 4 (males) and on lactation day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the WIL historical control data. Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL
historical control data ranges and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated during study week 4 (males) or on lactation day 4 (females).

ORGAN WEIGHTS
There were no test item-related alterations in final body weights or organ weights at the scheduled necropsies. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Lower thyroid/parathyroid gland weights relative to body weight were noted in the 500 mg/kg/day group females at the primary necropsy. A dose response was lacking and the difference was not attributed to the test item. Higher adrenal gland weights were noted in the 1000 mg/kg/day group males (relative to body weight) and females (relative to brain weight) at the recovery necropsy. There were no differences noted in adrenal gland weights at the primary necrospy and the differences in weights were of minimal magnitude; therefore, the
findings were attributed to biological variability. Lower right epididymides weights relative to brain weight were noted in the 1000 mg/kg/day group males at the recovery necropsy. The difference was unlilateral, was not noted at the primary necropsy, and epididymis and brain weights lack a proportional relationship (Bailey et al., 2004). A higher spleen weight relative to brain weight was noted in the 1000 mg/kg/day group females at the recovery necropsy. No differences in spleen weights were noted at the primary necropsy, and spleen and brain weights lack a proportional relationship (Bailey et al., 2004).

GROSS PATHOLOGY
No test item-related internal findings were observed at any dosage level in males and females at the scheduled necropsies. Macroscopic findings observed in the test item-treated groups occurred infrequently, similarly in the control group, and/or in a manner that was not dose-related.
The mean numbers of implantation sites in the 100 and 350 mg/kg/day groups were significantly (p<0.05) higher than the control group. These values were within the WIL Research historical control data range and in the absence of a dose-response were not considered to be test item-related. The mean numbers of implantation sites in the 500 and 1000 mg/kg/day groups and unaccounted-for sites in the 100, 350, 500, and 1000 mg/kg/day groups were similar to the control group values.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test item-related histologic changes noted at the primary necropsy. Histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
1000 mg/kg/day
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of pups born, live litter size and the percentage of males at birth in the 100, 350, 500, and 1000 mg/kg/day groups were similar to the control group values. Postnatal survival in the 100, 350, 500, and 1000 mg/kg/day groups were unaffected by test item administration.

CLINICAL SIGNS (OFFSPRING)
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental administration of the test item. Pups (litters) that were found dead numbered 19(3), 2(2), 4(3), 4(3), and 1(1) in the control, 100, 350, 500, and 1000 mg/kg/day groups, respectively. One, 1, 3, and 3 pups in the control, 100, 350, and 500 mg/kg/day groups, respectively, was missing and presumed to have been cannibalized.

BODY WEIGHT (OFFSPRING)
Mean male and female pup body weights and body weight changes during PND 1-4 in the 100, 350, 500, and 1000 mg/kg/day groups were unaffected by parental administration of the test item. No statistically significant differences from the control group were noted.

GROSS PATHOLOGY (OFFSPRING)
The numbers of litters found dead during PND 0-4 numbered 19(3), 2(2), 4(3), 4(3), and 1(1) in the control, 10, 350, 500, and 1000 mg/kg/day groups, respectively. The number of pups found dead in the control group was due to the total litter loss of female no. 2656 resulting from pup drowning due to a flooded cage. Pup no. 2683-01 in the 350 mg/kg/day group had fused kidneys.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

REPRODUCTIVE PERFORMANCE

F0 male and female reproductive parameters are presented in the following table.

 

Text Table 2. Results ofReproductive Performance

 

 

Parameter

 

0

DosageLevel(mg/kg/day)100          350         500

 

1000

WILHCa

Mean(Range)

MaleMatingIndex

100.0

100.0

100.0

100.0

100.0

95.9(86.7-100.0)

FemaleMatingIndex

100.0

100.0

100.0

100.0

100.0

96.6(86.7-100.0)

MaleFertilityIndex

100.0

100.0

100.0

100.0

100.0

88.1(70.0-100.0)

FemaleFertilityIndex

100.0

100.0

100.0

100.0

100.0

88.6(70.0-100.0)

MaleCopulationIndex

100.0

100.0

100.0

100.0

100.0

92.6(70.0-100.0)

FemaleConceptionIndex

100.0

100.0

100.0

100.0

100.0

92.6(70.0-100.0)

Pre-CoitalInterval(days)

a=WILhistoricalcontroldata

2.6

2.6

2.0

2.6

2.7

3.0(1.8-4.7)

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, no test item-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 100, 350, 500, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.
Executive summary:

OBJECTIVE

This study was designed to evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development.

STUDY DESIGN

The test item in the vehicle (arachis [peanut] oil) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 100, 350, 500, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-45 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 39 doses for females, these animals were assigned to a 15-day non-dosing recovery period.

All animals were observed twice daily for mortality and morbidity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on 5 F0 animals/sex/group at the primary necropsy and from 5 animals/sex in the control and high-dose groups at the recovery necropsy. F0 males were euthanized following completion of the mating period or 15-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered or following the 15-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups at the primary necropsy.

 

RESULTS

All F0 males and females in the control, 100, 350, 500, and 1000 mg/kg/day groups survived to the scheduled necropsies. Test item-related clinical findings noted for males and females in all test item-treated groups consisted of clear and/or red material around the mouth and/or red material around the nose approximately 1 hour following dose administration. These findings did not persist to the daily examinations on the following days and therefore were not considered adverse. In addition, clinical findings noted in the 1000 mg/kg/day group during the recovery period were limited to scabbing on the trunk and hair loss and scabbing on the facial area for F0 males and females, respectively.

No test item-related effects on F0 body weight or food consumption parameters were noted for males and females at any dosage level. There were no test item-related effects noted during the FOB or motor activity evaluations for F0 animals at any dosage level.

Mean male and female mating, fertility, copulation, and conception indices in the 100, 350, 500, and 1000 mg/kg/day groups were unaffected by test item administration. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration. There were no test item-related gross necropsy observations, microscopic findings, or effects on organ weights noted for F0 males and females at any dosage level during the dosing and recovery periods. No test item adverse alterations in clinical pathology parameters (hematology, coagulation, and serum chemistry) were noted for F0 males and females in the 100, 350, 500, and 1000 mg/kg/day groups during the dosing and recovery evaluations.

Mean numbers of former implantation sites and F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, and pup body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test item administration. Necropsy findings for F1 pups that died were not suggestive of any association with maternal administration of the test item.

 

CONCLUSION

Under the conditions of this screening study, no test item-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 100, 350, 500, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.