Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 September 2016 to 27 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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:
yes
Remarks:
various deviations with no impact on results or integrity of the study (see attached document)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Light brown, slightly viscous, liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Overall design of the study is shown in the diagram attached.
- Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test
system on this study. The animal model, the Crl:CD(SD) rat, is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, Charles River Ashland has reproductive historical control data in the
Crl:CD(SD) rat.
- The number of animals selected for this study (15 [Groups 1 and 5] or 10 [Groups 2–4]) rats/sex/group was based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group.
- Females were evaluated for oestrous cyclicity during the pre-test period and any females that failed to exhibit normal 4–5 day oestrous cycles (e.g. EDDE), repeatedly during the pre-test period were excluded from the study, therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.
- Crl:CD(SD) rats (66 males and 78 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 27 Sep 2016. The animals were approximately 54 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. On the day following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for an acclimation period of 21 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behaviour, and the testes were palpated at least once for all males.

ANIMAL HOUSING
- Following receipt and until pairing, all F0 animals were housed 2–3/cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs; The Andersons, Cob Products Division, Maumee, OH). The bedding material is periodically analysed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at Charles River.
- 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 solid-bottom cages until the scheduled necropsy. Following positive evidence of mating, the females were individually housed in solid-bottom cages with bedding material. The dams and their litters were housed in these cages until euthanasia on Lactation Day 13.
- 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.
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory
Animals.2 The animal facilities at Charles River Ashland are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitised weekly.

DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River. Feed lots used during the study were documented in the study records. Feeders were changed and sanitised once per week.
- Municipal water supplying the facility was sampled for contaminants according to Charles River SOPs. The results of the diet and water analyses are maintained at Charles River. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study, with the following exception. All F0 males and females (including animals not selected for clinical pathology evaluation) were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

ENVIRONMENTAL CONDITIONS
- All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 68 to 78 °F (20 to 26 °C) and 30 % to 70 %, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 66.3 °F to 72.6 °F (19.1 °C to 22.6 °C) and mean daily relative humidity ranged from 27.6 % to 48.1 % during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Lot 2EL0356, expiry date 31 December 2016 (test substance formulations); Lot 2FG0172, expiry date 30 June 2017 (control group)
Details on oral exposure:
TEST ITEM PREPARATION
- The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature (18 °C to 24 °C), protected from light. The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.
- The test substance formulations (see table below) were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18 °C to 24 °C), protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- During the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to
be in good health, meeting acceptable body weight requirements, and exhibiting normal 4–5 day oestrous cycles (females) was selected for use in the computerized randomization procedure based
on body weight stratification in a block design. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated. The animals then were arranged into groups according to the printout. Animals not assigned to study were transferred to the Charles River rat colony.
- The experimental design consisted of 4 test substance-treated groups and 1 control group composed of 10 rats/sex/group. An additional 5 rats/sex in the control and high-dose group were
selected to be evaluated following a 15-day recovery period; animals assigned to the recovery period were not evaluated for reproductive toxicity. At the initiation of dose administration (Study Day 0), the males and females were approximately 11 weeks old. Male body weights ranged from 337 g to 435 g and female body weights ranged from 209 g to 280 g on Study Day 0. The animals were approximately 13 weeks old when paired on Study Day 13; female body weights ranged from 222 g to 309 g on Gestation Day 0.

TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- Study group assignment is shown in the table below.
- The vehicle and test substance formulations were administered orally by gavage, via appropriately sized flexible, disposable, plastic feeding tubes (Instech Solomon, Plymouth Meeting, PA) once daily. - 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 13) for a total of 49-56 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 49 doses for females, these animals remained on study for a 15-day recovery period.
- The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
- Dosage levels were selected based on the results of a previous 14-day range-finding study3 in which male and female rats were administered the test substance at dosage levels of 100, 300, 500, and 1000 mg/kg/day. There were no significant clinical observations noted in any of the dosage groups. In addition, mean body weights and food consumption were unaffected by treatment. As a result, the same dosage levels were chosen for the current study.
- The selected route of administration for this study was oral (gavage) because this a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSIS
- Homogeneity of the test substance in the vehicle and resuspension homogeneity and stability following 6 and 10 days of room temperature storage at concentrations of 10 and 220 mg/mL were established in a previous study. Therefore, stability assessments were not conducted in the current study.
- Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first and last test substance dosing formulations and from the middle stratum of the first and last control group dosing formulations. The 20 mg/mL formulation prepared on 05 Dec 2016 did not meet acceptance criteria and was reformulated on 06 Dec 2016; samples for homogeneity and concentration determinations were collected from the top, middle, and bottom strata of this formulation and were found to be within the acceptable concentration range. One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored at room temperature as back-up. All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.
Duration of treatment / exposure:
- 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 13 for a total of 49–56 doses.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- Low- and mid-dose groups (Groups 2–4):10 rats/sex
- High-dose group (Group 5):15 rats/sex
- Concurrent control group: 15 rats/sex
Control animals:
yes, concurrent vehicle
Details on study design:
DATA ACQUISITION AND ANALYSIS
- The major computer systems used on this study include, but are not limited to, the systems shown in the table attached.
- All computerised systems used for data collection during the conduct of this study have been validated (with the exception of Microsoft Office); when a particular system has not satisfied all requirements, appropriate administration and procedural controls were implemented to assure the quality and integrity of the data.

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.
- Individual clinical observations were recorded daily and 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. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.
- Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labour, delayed labour) or other difficulties. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal; only positive findings were recorded.

BODY WEIGHTS
- Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia.
- Individual female body weights were recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase).
- Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating dosing period (males and females) and for the entire dosing period (males and recovery phase females).
- Once evidence of mating was 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, 4, 7, 10, 13, and 14 (fasted; see Appendix 1 - Study Protocol and Deviations). Body weight changes were presented for each of these intervals and for the entire gestation and lactation intervals (Days 0– 20 and 1–13, respectively).
- Weekly body weights for females with no evidence of mating were presented on the individual report tables until necropsy.
- When body weights could not be determined for an animal during a given interval (as females enter gestation, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables.

FOOD CONSUMPTION
- 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 as 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, 4, 7, 10, and 13.
- Following the breeding period, individual food consumption for females with no evidence of mating and for all males was measured on a weekly basis until the scheduled euthanasia.
- Weekly food consumption for females with no evidence of mating was presented in the individual report tables until necropsy.
- Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.
- When food consumption could not be determined for a given interval (due to a weighing error, scheduled euthanasia, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable were left blank or designated as “NA” on the individual report tables.

FOB ASSESSMENTS
- All animals were observed for the parameters listed in the table below.
- FOB assessments were recorded for 5 animals/sex/group following approximately 28 days of dose administration (Study Week 3, males selected for pairing) or on Lactation Day 13 (females). The FOB used at Charles River is based on previously developed protocols.
- FOB testing was performed by the same biologists, to the extent possible, without knowledge of the animal’s group assignment. The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB.
- Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer. The animal was allowed to grip a T-shaped grip bar with its forepaws and was pulled back gently along a platform until its grip was broken. As the backward locomotion continues, the animal’s hindpaws reach a T-shaped rearlimb grip bar, which it is allowed to grasp and then forced to release by continued pulling. Mark-10 series EG digital force gauges (Mark-10 Corporation, Copiague, NY) were used to record the maximum strain required to break forelimb and hindlimb grip. The average of 3 valid measurements was taken as the animal’s score for each grip strength measure.

MOTOR ACTIVITY
- Motor activity was assessed for 5 animals/sex/group following approximately 28 days of dose administration (Study Week 3, males selected for pairing) or on Lactation Day 13 (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. Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams.
- The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 60 dB to 80 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. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY
- Parameters listed in the tables below were evaluated.
- Blood samples for clinical pathology evaluations (haematology, coagulation, and serum chemistry) were collected from 5 animals/sex/group at the scheduled/primary necropsies (Study Day 28 for males and Lactation Day 14 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 (reported as Study Week 6) for males and Study Day 63 (reported as Study Week 9) for females. The animals were fasted overnight prior to blood collection.
- Blood for serum chemistry and haematology 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. Blood was collected into tubes containing K2EDTA (haematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).


Sacrifice and pathology:
MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals at the scheduled termination.
- Tissue collection was as shown in the table below.
- All F0 adults were euthanised by carbon dioxide inhalation.
- Males were euthanised following completion of the mating period or following the 15-day recovery period; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.
- Females that delivered were euthanised on Lactation Day 14; blood samples were collected for
thyroid hormone analysis immediately prior to euthanasia. For females that delivered, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female that delivered by subtracting the number of pups born from the number of former implantation sites observed. Females not selected for pairing were euthanized following the 15-day recovery period.
- Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. At the time of necropsy, the following tissues and organs were placed in 10% neutral buffered formalin (except as noted).

ORGANS WEIGHED AT NECROPSY
- Except as noted in the table below, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported.
- When organ weights could not be determined for an animal (due to weighing error, lost or damaged organ, etc.), group mean values were calculated using the available data. The organs for which weights could not be determined were designated as “NA” on the individual report tables.

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
- After fixation, protocol-specified tissues were trimmed according to Charles River SOPs and the
protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to Charles River SOPs, mounted on glass microscope slides, and stained with haematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
- Microscopic examination was performed on all tissues from all animals in the control and 1000 mg/kg/day groups at the primary necropsy. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate (see Appendix 1 - Study Protocol and Deviations). Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.


Statistics:
See below

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
- No test substance-related clinical observations were noted for the 100, 300, 500, and 1000 mg/kg/day groups at the daily examinations, weekly detailed physical examinations, or approximately 1 hour following dose administration.
- Findings noted in the test substance-treated groups, including red material around the nose and mouth and hair loss on the forelimbs, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mortality:
no mortality observed
Description (incidence):
- All F0 males and females survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Males: Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group F0 males were unaffected by test substance administration throughout the study. Mean body weight gain in the 1000 mg/kg/day group was significantly (p < 0.05) lower than the control group during Study Days 21–27. In the absence of a corresponding effect on mean body weights, the transient difference in mean body weight gain in the 1000 mg/kg/day group was not considered test substance-related. No other statistically significant differences from the control group were noted. During the recovery period (Study Days 28–41) mean body weights and body weight gains in the 1000 mg/kg/day group were similar to the control group.
- Females (weekly): Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group F0 females were unaffected by test substance administration during the pre-mating period (Study Days 0–13). None of the differences from the control group were statistically significant. Mean body weight gain for the 1000 mg/kg/day group F0 females not paired for mating was similar to the control group for the remainder of the dosing period (Study Days 13–48). However, a significantly (p < 0.05) higher mean body weight gain was noted for these females compared to the control group when the overall dosing period (Study Days 0–48) was evaluated. This difference was not of sufficient magnitude to affect mean body weights in this group and therefore was not considered test substance-related. During the recovery period (Study Days 49–62), mean body weights and body weight gains in the 1000 mg/kg/day group F0 females were similar to the control group; differences from the control group were slight and not statistically significant.
- Females (gestation): Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group F0 females were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group F0 females were unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Males: Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group F0 males was similar to that in the control group throughout the study. No statistically significant differences were observed.
- Females (weekly): Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group F0 females was unaffected by test substance administration during the pre-mating period (Study Days 0–13). Differences from the control group were slight and not statistically significant, with the following exception. Significantly (p < 0.01) higher mean food consumption was noted for the 300 mg/kg/day group during Study Days 0–7. In the absence of a dose-response, this transient increase in mean food consumption was not considered test substance related. Mean food consumption for the 1000 mg/kg/day group F0 females not paired for mating was similar to the control group for the remainder of the dosing period (Study Days 27–48); the differences were slight and not statistically significant. During the recovery period (Study Days 49–62), mean food consumption in the 1000 mg/kg/day group was similar to the control group.
- Females (gestation): Mean maternal food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day groups was unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean maternal food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no test substance-related effects on haematology and coagulation parameters.
- The only significant (p < 0.05) difference from the control group was a higher mean platelet count in the 1000 mg/kg/day group F0 males at the recovery necropsy. However, the value was within the Charles River Ashland historical control data and there was no effect on any correlating parameter at the primary necropsy; therefore, this increase was not considered test substance-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
SERUM CHEMISTRY
- There were no test substance-related effects on serum chemistry. Significant (p < 0.05) differences from the control group at the primary necropsy included higher mean albumin and total protein levels in the 300 and 1000 mg/kg/day group F0 males, higher mean alanine aminotransferase (ALT) and cholesterol values in the 100 mg/kg/day group F0 males, and a higher mean aspartate aminotransferase (AST) value in the 100 mg/kg/day group F0 females. At the recovery necropsy, a significantly (p < 0.05) higher mean alkaline phosphatase (ALP) value was noted in the 1000 mg/kg/day group F0 males compared to the control group. There was no dose-response relationship, no similar occurrence in the opposite sex, no histopathologic correlates, and/or the changes were of minimal magnitude. Therefore, the differences from the control group during the dosing and recovery evaluations were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- Home cage parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).
- Handling parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 or on Lactation Day 13 (females).
- Open field parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).
- Sensory parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).
- Neuromuscular parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).
- Physiological parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no test substance-related alterations in organ weights at the scheduled necropsies. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. At the primary necropsy, lower mean seminal vesicle/coagulating gland weights were noted in the 300 (absolute and relative to final body weight) and 1000 (absolute) mg/kg/day group males. Lower weight correlated macroscopically with small seminal vesicle and microscopically with decreased secretion in a single 300 mg/kg/day group male. A dose-response relationship was lacking and individual values were above the lower limit of the historical control database; the difference was attributed to biological variability.
- At the recovery necropsy, higher mean seminal vesicle/coagulating gland (relative to brain weight) and adrenal gland (relative to final body and brain weights) weights were noted in the 1000 mg/kg/day group males, and higher mean pituitary gland weights (absolute and relative to final body and brain weights) were noted in the 1000 mg/kg/day group females. Higher organ weights were not noted at the primary necropsy and weights were within the historical control database range; the findings were attributed to biological variability.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.
- The mean numbers of unaccounted-for sites and implantation sites in the 100, 300, 500, and 1000 mg/kg/day groups were similar to the control group values.
Neuropathological findings:
no effects observed
Description (incidence and severity):
MOTOR ACTIVITY
- Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all concentrations when evaluated during Study Week 3 (males) and on Lactation Day 13 (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 Charles River Ashland historical control data.
- Differences from the control group were slight, not statistically significant when analysed by a repeated measures analysis, within the Charles River Ashland 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 3 (males) and on Lactation Day 13 (females).
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
MICROSCOPIC EXAMINATIONS
- No test substance-related microscopic findings were noted.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES
- There were no test substance-related effects on thyroid hormone values in the F0 males at any dosage level. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: equivalent to > 590 mg active ingredient/kg bw/day

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

ANALYSES OF DOSING FORMULATIONS

- Results of the analyses of dosing formulations are summarised in the table attached.

- The analysed dosing formulations were within Charles River SOP range for suspensions (85% to 115%) and the protocol-specified requirement for homogeneity.

- The test substance was not detected in the analysed vehicle formulation that was administered to the control group (Group 1).

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, based on the lack of any test substance-related effects at any dosage level, a dosage level of 1000 mg/kg/day (the highest dosage level evaluated) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity, F0 systemic toxicity, and F1 neonatal toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. The actual dosage levels based on the percentage active ingredient listed on the revised Certificate of Analysis received during the study were 59, 177, 295, and 590 mg/kg/day.
Executive summary:

GUIDELINE

The objectives of the study were to investigate the potential toxic effects of the test substance when administered to rats for a minimum of 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behaviour, and conception through day 13 of postnatal life. The protocol was designed in general accordance with the OECD Guideline for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016.

 

METHODS

The test substance, 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 24) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 100, 300, 500, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Males and females were approximately 11 weeks of age at the beginning of test substance 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 13 for a total of 4956 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 49 doses for females, these animals were assigned to a 15-day non-dosing recovery period.

 

All animals were observed twice daily for mortality and moribundity. 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 13. All F0 females selected for pairing were allowed to deliver and rear their pups until Lactation Day 13. F1 clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (2/litter). All F1 male pups were evaluated for areolae/nipple anlagen on PND 13. Remaining F1 pups were euthanized on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (haematology, coagulation, and serum chemistry) were performed on 5 F0 animals/sex/group at the primary necropsy and 5 animals/sex in the control and high-dose groups at the recovery necropsy. Blood samples for thyroid hormone analysis were collected from F0 males and females at the primary (Lactation Day 14 for females) and recovery necropsies; only primary necropsy male samples were analysed. F0 males were euthanised following completion of the mating period or 15-day recovery period and F0 females were euthanized on Lactation Day 14 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 survived to the scheduled necropsies. No test substance-related clinical observations were noted for F0 males and females in the 100, 300, 500, and 1000 mg/kg/day

groups at the daily examinations, weekly detailed physical examinations, or approximately 1 hour following dose administration. No test substance-related effects were noted on parental body weights, body weight gains, or food consumption at any dosage level throughout the study. Mean F0 male and female reproductive performance (mating, fertility, and copulation/conception

indices) and the process of parturition in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test substance administration. The mean number of days between pairing and coitus, gestation length, and oestrous cycle length were unaffected by test substance administration. There were no test substance-related effects noted on the mean numbers of implantation sites and unaccounted-for sites in F0 females at any dosage level. There were no test substance-related effects noted during the FOB or motor activity evaluations for F0 males and females at any dosage level.

 

There were no test substance-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 substance-related alterations in clinical pathology parameters (haematology, coagulation, and serum chemistry) were noted for F0 males and females in the 100, 300, 500, and 1000 mg/kg/day groups during the dosing and recovery evaluations. Mean serum T4 levels in the F0 males were unaffected by test substance administration. Mean numbers of F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, pup body weights, and body weight gains, anogenital distance, and areolae/nipple anlagen (males only) in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by parental test substance administration. No necropsy findings were noted for F1 pups in the test substance-treated groups on PND 13. There were no test substance-related changes in mean serum T4 levels or mean thyroid/parathyroid weights in F1 males and females on PND 13.

 

CONCLUSION

 

Under the conditions of this screening study, based on the lack of any test substance-related effects at any dosage level, a dosage level of 1000 mg/kg/day (the highest dosage level evaluated) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity, F0 systemic toxicity, and F1 neonatal toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. The actual dosage levels based on the percentage active ingredient listed on the revised Certificate of Analysis received during the study were 59, 177, 295, and 590 mg/kg/day.