Registration Dossier

Administrative data

Description of key information

Key Study 1: Under the conditions of a screening study involving an analogue test material, 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 30, 100, 250, 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 (OECD 422).

Key Study 2: Administration of an analogue test item via oral gavage to Sprague Dawley rats for 28 consecutive days was well tolerated at dosage levels of 30, 100, 250, and 1000 mg/kg/day and resulted in non-adverse clinical observations in the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day (the highest dosage level tested) (OECD 407).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2015 to 22 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Remarks:
various deviations with no impact on results or integrity of the study (see Appendix A, attached)
GLP compliance:
yes
Limit test:
no
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
- Crl:CD(SD) rats were used as the test system for this study. This species and strain of animal is recognized as appropriate for subchronic oral (gavage) toxicity studies. The Sprague Dawley rat was selected because it is a widely used strain for which significant historical control data are available. The number of animals selected for this study was the minimum necessary to yield scientifically meaningful results based on the OECD Guidelines for Testing of Chemicals: Guideline 407.
- Crl:CD(SD) rats (40 males and 40 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 20 January 2015. The animals were approximately 5 weeks old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed 3 days later. Each animal was uniquely identified with a subcutaneous microchip (BMDS system) implanted in the dorsoscapular area. All animals were housed for a 14-day acclimation period. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior. Data collection during acclimation began on 23 January 2015. Individual body and food weights were recorded and detailed physical examinations were performed periodically during acclimation.

ANIMAL HOUSING
- Upon arrival, all animals were housed 2 to 3 per cage by sex in clean, solid-bottom cages containing ground corncob bedding material (Bed O’Cobs).
- The bedding material is periodically analysed by the manufacturer for contaminants. Analyses of the nesting material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the objectives of this study. The results of these analyses are maintained at WIL Research.
- The animals were temporarily separated as necessary to allow for the performance of protocol-specified activities. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research 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 sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research.
- Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinical pathology blood collection and necropsy when food, but not water, was withheld.
- Municipal water supplying the facility was analysed for contaminants according to the WIL Research SOPs. The results of the diet analyses are maintained in the study records, and the results of the water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.
- In order to maintain the health status of individual animals during the course of the study, one social group was offered a water bottle on study day 21.

ENVIRONMENTAL CONDITIONS
- All animals were housed throughout acclimation and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71 ± 5 °F (22 ± 3 °C) and 50 ± 20 %, 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 68.1 °F to 71.9 °F (20.1 °C to 22.2 °C) and mean daily relative humidity ranged from 25.1 % to 48.3 % 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- On 28 January 2015 (6 days prior to the initiation of dose administration), all available rats were weighed and examined in detail for physical abnormalities. These data were collected using WTDMS and reviewed by the Study Director. The animals judged suitable for assignment to the study were selected for use in a computerized randomisation procedure based on body weight stratification in a block design. A printout containing the animal numbers and individual group assignments was generated, and the animals were then arranged into groups according to the printout. Individual body weights at randomisation were within ± 20 % of the mean for each sex. Animals not assigned to study were transferred to the WIL Research colony.
- The control and 1000 mg/kg/day groups (Groups 1 and 5, respectively) each consisted of 10 males and 10 females, and the 30, 100, and 250 mg/kg/day groups (Groups 2, 3, and 4, respectively) each consisted of 5 males and 5 females. These animals were then randomised into 3 study replicates to allow for the reasonable conduct of the functional observational battery and locomotor activity assessments. Each dose group and sex was approximately equally represented within each study replicate. The animals were approximately 7 weeks old at the initiation of dose administration. Individual body weights ranged from 186 g to 225 g for males and from 143 g to 180 g for females at randomisation.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Lot numbers 2EA0218 and 2EA0347; Expiry date 31 January 2016
Details on oral exposure:
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 refrigerated (2 °C to 8 °C), protected from light. The vehicle was mixed throughout the sampling and dose administration procedures.
- Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, 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.

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible Teflon-shafted, stainless steel ball-tipped dosing cannula once daily for 28 consecutive days, through the day prior to the primary necropsy. The dose volume for all groups was 5 mL/kg. Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dosage. Adjusted doses became effective the day of collection of the weekly body weights. All animals were dosed at approximately the same time each day. The first day of dosing was study day 0; the first week of dosing was study week 0.
- Dosage levels were determined using the results of a previous study conducted orally in rats (Consumer Products Testing, Study No. 82466, 1983) showing a median lethal dosage (LD50) of >5000 mg/kg with this test substance. It was anticipated that the high-dosage level would show drug-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation. The lower dosage levels were selected at intervals that were predicted to be narrow enough to reveal any dose related trends.
- The selected route of administration for this study was oral (gavage).
- Study group assignment is shown in the table below.

DATA ACQUISITION, ANALYSIS AND REPORTING
- The major computer systems used on this study include, but are not limited to systems listed in the attached table.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSES
- The test substance in the vehicle was found to be stable at concentrations of 5 and 230 mg/mL following 8 days of refrigerated storage in a previous study (Bystry, 2015, WIL-168232).
- Samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the first 6, 20, 50, and 200 mg/mL dosing formulations prepared for dose administration on study day 0. In addition, aliquots of sufficient volume for dosing a group of animals for 1 day were prepared and stored at room temperature, protected from light, for 8 days.
- The representative aliquots were then mixed with a magnetic stirrer for a minimum of 30 minutes, and samples were collected from the top and bottom strata of each aliquot for resuspension homogeneity determinations. In addition, samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared for the last week (study week 3) of dose administration. One duplicate set was analysed and the remaining duplicate set was stored refrigerated and retained as backup samples.
- All analyses were conducted by the WIL Research Analytical Chemistry Department using a method validated on a previous study (Bystry, 2015, WIL-168232).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- Vehicle control: 10 males and 10 females
- Test item administered at 30, 100 and 250 mg/kg bw/day: 5 males and 5 females
- Test item administered at 1000 mg/kg bw/day: 10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- A flow chart summarising the study design is attached.
Observations and examinations performed and frequency:
SURVIVAL
- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

CLINICAL OBSERVATIONS
- Clinical examinations were performed at the time of dose administration and 1-2 hours following dose administration. During the recovery period, the animals were observed once daily. The absence or presence of findings was recorded for individual animals at the scheduled intervals.
- Detailed physical examinations were conducted on all animals 1 week (± 2 days) prior to randomisation, on the day of randomisation, weekly (± 2 days) during the study period, and on the days of the scheduled necropsies.
- Due to social housing, some observations could not be attributed to a single animal. In these instances, the observation was recorded in a separate computer protocol for the social group.

BODY WEIGHTS
- Individual body weights were recorded 1 week (± 2 days) prior to randomisation, on the day of randomisation, on study day 0 (prior to dosing), weekly (± 2 days) during the study period, and on the day prior to the scheduled necropsies (non-fasted).
- Mean body weights and mean body weight changes were calculated for the corresponding intervals.
- In addition, individual body weights were recorded for male nos. 1775 and 1777 in the 1000 mg/kg/day group on study day 20.
- Final body weights (fasted) were recorded on the day of the scheduled necropsies.

FOOD CONSUMPTION
- Cage food weights were recorded once weekly (± 2 days) beginning following randomisation and throughout the study period. Food consumption was calculated and normalized to the number of animals/cage and reported as g/animal/day for the corresponding body weight intervals.
- When food consumption could not be measured for a given interval (due to weighing error, obvious erroneous value, etc.), the appropriate interval was footnoted as "NA" on the individual tables.

FOB ASSESSMENTS
- FOB assessments were recorded for 5 animals/sex/group during the final week of test substance administration (study week 3) and on all remaining animals during the final week of the recovery period (study week 5).
- Testing was performed by the same technicians, whenever possible, without knowledge of the animals’ group assignments, and was performed at approximately the same time each day.
- The FOB was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. The FOB used at WIL Research is based on previously developed protocols (Moser et al., 1991; Irwin, 1968; Gad, 1982; Moser et al., 1988; Haggerty, 1989; O'Donoghue, 1989).
- All animals were observed for the parameters listed in the table below.

LOCOMOTOR ACTIVITY
- Locomotor activity, recorded after the completion of the FOB, was assessed for 5 animals/sex/group during the final week of test substance administration (study week 3) and on all remaining animals during the final week of the recovery period (study week 5).
- Locomotor activity was measured automatically using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding a clear, plastic rectangular cage to quantify an animal’s locomotor activity.
- Four-sided black plastic enclosures were used to surround the clear plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by technicians or adjacent
animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The locomotor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB.
- The testing of treatment groups was conducted according to replicate sequence. 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 sub- sessions for tabulation.
- Data for ambulatory and total locomotor activity were tabulated. Total locomotor activity was defined as a combination of fine locomotor skills (i.e., grooming; interruption of a single photobeam) and ambulatory locomotor activity (e.g., interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY
- Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected at the scheduled necropsies (study days 28 and 42) from animals scheduled for necropsy.
- The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection.
- Blood was collected at the time of euthanasia via the inferior vena cava of animals anesthetized by inhalation of isoflurane. Blood was collected into tubes containing potassium (K2) EDTA (haematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

HAEMATOLOGY AND COAGULATION
- The parameters listed in the table below were examined.

SERUM CHEMISTRY
- The parameters listed in the table below were examined.

URINALYSIS
- The parameters listed in the table below were examined.
Sacrifice and pathology:
ANATOMIC PATHOLOGY – MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all animals. Animals were anesthetized by isoflurane inhalation and euthanized by exsanguination.
- The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
- Except where noted, the tissues and organs listed in the table below were collected and placed in 10 % neutral-buffered formalin.

ORGAN WEIGHTS
- Organs listed in the table below were weighed from all animals at the scheduled necropsies.
- Paired organs were weighed together.
- The organ to final body weight and organ to brain weight ratios were calculated.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION
- After fixation, protocol-specified tissues were trimmed according to the WIL Research SOPs and the protocol. Trimmed tissues with gross lesions from all groups and all tissues from animals in the control and 1000 mg/kg/day group were processed into paraffin blocks, sectioned according to the WIL Research SOPs, mounted on glass microscope slides, and stained with haematoxylin and eosin.
- Microscopic examination was performed on tissues collected from all animals in the control and 1000 mg/kg/day groups at the primary necropsy and on all gross lesions from all groups.
Statistics:
See below
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related clinical observations were noted for the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females during the dosing period.
- Test substance-related increased incidences of red material around the nose were noted for the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females during the dosing period. In addition, test substance-related clear material around the mouth was noted for the 1000 mg/kg/day group males and females, and clear material around the ventral neck was noted for the 1000 mg/kg/day group females. These clinical findings were primarily noted at the time of dosing and/or 1-2 hours post-dosing and generally did not persist to the recovery period.
- All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
- All animals survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Body weights were unaffected by test substance administration.
- There were no statistically significant differences when the control and test substance-treated groups were compared, with the exception of a single statistically significantly lower mean body weight gain noted for the 250 mg/kg/day group males during study days 14-21 compared to the control group; this change was transient, did not occur in a dose-related manner, and was not considered test substance-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Food consumption was unaffected by test substance administration.
- A statistically significantly lower mean food consumption value was noted for the 1000 mg/kg/day group females during study days 35-41 (recovery period) when compared to the control group; however, the difference from the control group was slight and no test substance-related effects on food consumption were noted during the dosing period. Therefore, this change was not considered test substance-related. There were no other statistically significant differences when the control and test substance-treated groups were compared.
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 or coagulation parameters.
- However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Mean absolute neutrophil and monocyte counts were higher in the 1000 mg/kg/day group males at the recovery necropsy, but these values were within the range of mean values in the WIL Research historical control database.
- Additionally, there were no alterations in these parameters in the test substance-treated groups at the primary necropsy; thus, these alterations were considered to be due to biologic variability. Statistically significant findings that involved percentage leukocyte differential counts were not itemised above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no test substance-related effects on serum chemistry parameters.
- However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Mean aspartate aminotransferase was higher in the 250 mg/kg/day group males at the primary necropsy, but this level was within the range of mean values in the WIL Research historical control database.
- Additionally, there was no alteration in this parameter in the 1000 mg/kg/day group at either the primary or recovery necropsies; thus, this alteration was considered to be due to biologic variability. The mean glucose level was lower in the 1000 mg/kg/day females at the recovery necropsy; however, the level was within the WIL Research historical control database. Additionally, there was no alteration in this parameter in the primary test substance-treated groups; thus, this alteration was considered to be due to biologic variability.
Urinalysis findings:
no effects observed
Description (incidence and severity):
- There were no test substance-related alterations in urinalysis parameters.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Home cage observations: Home cage observations were unaffected by test substance administration. A statistically significantly lower number of 250 mg/kg/day group females was noted as sitting or standing normally when compared to the control group at the study week 3 evaluation, and corresponding higher (not statistically significant) numbers of 250 mg/kg/day group females were noted as alert and oriented toward the observer or asleep, lying on side, or curled up. However, the postures noted for the 250 mg/kg/day group females are common for rats. In addition, no corresponding posture-related clinical observations were noted that would suggest a test substance-related effect and a similar change in
posture was not observed for the 1000 mg/kg/day group females. Therefore, these changes were not considered test substance-related. There were no other statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 and 5 evaluations.
- Handling observations: Handling observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 and 5 evaluations.
- Open field observations: Open field observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 and 5 evaluations.
- Sensory observations: Sensory observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 and 5 evaluations.
- Neuromuscular observations: Neuromuscular observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 and 5 evaluations.
- Physiological observations: Physiological observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 and 5 evaluations.
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 effects on organ weights.
- However, the following statistically significant differences were observed when the control and test substance-treated groups were compared. The mean liver/brain weight ratio was higher in the 1000 mg/kg/day group females at the primary necropsy compared to the control group; however, the ratio was within the range of the WIL Research historical control database. Therefore, this apparent weight ratio variation was considered to be due to biologic variation.
- The mean epididymides/body weight ratio was higher in the 1000 mg/kg/day group males at the recovery necropsy compared to the control group; however, the ratio was within the range of the WIL Research historical control database, there were no notable histologic lesions in the treated animals, and no changes in the primary treated animals. Therefore, this apparent weight ratio variation was considered to be due to biologic variation.
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.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
MOTOR ACTIVITY
- Locomotor activity patterns (total and ambulatory activity counts) were unaffected by test substance administration. Values obtained from the 6 epochs evaluated (0-10 minutes, 11-20 minutes, 21-30 minutes, 31-40 minutes, 41-50 minutes, and 51-60 minutes) and the overall 60-minute test session were comparable to the concurrent control values, with the following exceptions.
- At the study week 3 evaluation, the mean total counts noted for the 30, 100, 250, and 1000 mg/kg/day group males (1090-1234 total counts) were similar to the mean control group value (1339 total counts) during the initial 0-10 minute subinterval. However, lower mean total counts were noted for males in the same groups during the subsequent 11-20, 21-30, 31-40, and 41-50 minute subintervals when compared to the control group; the differences were statistically significant for the 250 and 1000 mg/kg/day group males during the 11-20 and 31-40 minute subintervals. As a result of these changes, statistically significantly lower mean cumulative total counts were noted for the 100, 250, and 1000 mg/kg/day group males when the overall 60-minute session was evaluated compared to the control group. When rats are placed in a novel environment, the animals exhibit exploratory behavior that results in activity followed by habituation to the novel environment and reduced activity. Therefore, these differences from the control group were considered to be the result of faster habituation in the test substance-treated groups, a normal behavior in rats, and were not considered test substance-related.
- At the study week 5 recovery evaluation, a statistically significantly lower mean total count was noted for the 1000 mg/kg/day group females during the 11-20 minute subinterval when compared to the control group. This difference was due to faster habituation in this group and given the lack of test substance-related effects at the study week 3 evaluation, this change was not considered test substance-related.
- No other remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated on study weeks 3 and 5. There were no other statistically significant changes for the test substance-treated males and females when compared to the control group at the study week 3 and 5 evaluations.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no test substance-related histologic changes. All histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance.
- There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. A single female in the 1000 mg/kg/day recovery necropsy group had extensive pericardial and pleural fibrosis with chronic inflammation that extended into the mediastinum. This was not seen in any other treated animals and the cause was not apparent.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: study conducted in accordcance with OECD 407
Key result
Critical effects observed:
no

ANALYSES OF DOSING FORMULATIONS

- Summary tables showing results of homogeneity and concentration analyses are attached.

- The analysed dosing formulations contained 101 % to 111 % of the test substance, which was within the WIL Research SOP range of target concentrations for suspensions (85 % to 115 %) and were homogeneous immediately following preparation and following resuspension after 8 days of refrigerated storage. The test substance was not detected in the analysed vehicle formulations that were administered to the control group (Group 1).

Conclusions:
Based on the results of this study, administration of test item via oral gavage to Sprague Dawley rats for 28 consecutive days was well tolerated at dosage levels of 30, 100, 250, and 1000 mg/kg/day and resulted in non-adverse clinical observations in the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day (the highest dosage level tested).
Executive summary:

GUIDELINE

The study was conducted in general accordance with the OECD (2008), Test No. 407: Repeated Dose 28 Day Oral Toxicity Study in Rodents, OECD Guidelines for the Testing of Chemicals, Section 4. The objectives of the study were to evaluate the potential toxic effects of the test substance when administered via gavage to rats for 28 consecutive days, as well as to evaluate the recovery, persistence, or progression of any effects following a 14-day recovery period. This included evaluation of potential neurotoxicity by functional observational battery and motor activity assessment.

 

METHODS

The test substance, in the vehicle (peanut oil), was administered orally by gavage once daily for 28 consecutive days to 4 groups (Groups 2-5) of Crl:CD(SD) rats. Dosage levels were 30, 100, 250, and 1000 mg/kg/day for Groups 2, 3, 4, and 5, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Groups 1 and 5 each consisted of 10 animals/sex, and Groups 2-4 each consisted of 5 animals/sex. Following 28 days of dose administration, 5 rats/sex/group were euthanized; the remaining 5 rats/sex in the control and high-dose groups were euthanized following a 14-day non-dosing (recovery) period. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body and food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and locomotor activity data were recorded for 5 animals/sex/group during study week 3 and on all remaining animals during study week 5. Clinical pathology parameters (haematology, coagulation, serum chemistry, and urinalysis) were analysed for all animals scheduled for the primary (study day 28) and recovery (study day 42) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

 

RESULTS

All animals survived to the scheduled necropsy. Test substance-related clinical observations were noted during the dosing period and consisted of red material around the nose for the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females, clear material around the mouth for the 1000 mg/kg/day group males and females, and clear material around the ventral neck for the 1000 mg/kg/day group females. Recovery from these clinical observations was evident during the recovery period. There were no test substance-related alterations in body weights, food consumption, clinical pathology parameters, or organ weights. In addition, there were no test substance-related macroscopic or microscopic findings noted at the scheduled necropsies. No test substance-related neurobehavioral findings (FOB or motor activity alterations) were noted at the end of the dosing and recovery periods.

 

CONCLUSION

Based on the results of this study, administration of test item via oral gavage to Sprague Dawley rats for 28 consecutive days was well tolerated at dosage levels of 30, 100, 250, and 1000 mg/kg/day and resulted in non-adverse clinical observations in the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day (the highest dosage level tested).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 April 2015 to 11 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 Appendix A, attached)
GLP compliance:
yes
Limit test:
no
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
- 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 recognised as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, WIL Research 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 rats/sex/group [Groups 2-4]) was based on the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 22-Mar-1996, which recommends evaluation of at least 8 pregnant females per group.
- Given the possibility of non-gravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, 10 animals/sex/group was an appropriate number of animals to obtain a sample size of 8 at termination. In addition, 5 animals/sex in the control and high-dose
groups were necessary to assess recovery, persistence, or progression of any toxic effects following the cessation of dosing.
- Crl:CD(SD) rats (66 males and 66 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 28-Apr-2015. The animals were approximately 61 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. Two days 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 9 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 per 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 WIL Research.
- During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding material. Following the breeding period, the breeding phase males and 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 4. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until delivery or post-mating day 25. 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 (National Research Council, 2011). The animal facilities at WIL Research 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 oral health of the animals, and were sanitised weekly.

DIET, 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 WIL Research. 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 WIL Research SOPs. The results of the diet and water analyses are maintained at WIL Research. 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 males and recovery phase females 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 71 °F ± 5 °F (22 °C ± 3 °C) and 50 % ± 20 %, 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 70.3 °F to
72.5 °F (21.3 °C to 22.5 °C) and mean daily relative humidity ranged from 46.7 % to 66.4 % 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- At the conclusion of 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 and meeting acceptable body weight requirements was selected for use in the computerized randomisation 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 WTDMSTM. 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 WIL Research colony.
- The experimental design consisted of 4 test item-treated groups and 1 control group composed of 10 rats/sex/group. An additional 5 rats/sex in the control and high-dose groups were selected to be evaluated following a 14-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 10 weeks old. Male body weights ranged from 335 g to 414 g and female body weights ranged from 216 g to 265 g on study day 0. The animals were approximately 12 weeks old when paired on study day 13; female body weights ranged from 228 g to 288 g on gestation day 0.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Lot numbers 2EA0347 and 2EA0218; expiry date 31 January 2016
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 item formulations; aliquots were prepared for daily dispensation to the control group and stored refrigerated (2 °C to 8 °C), protected from light. The vehicle was mixed throughout the sampling and dose administration procedures. On each dosing day, aliquots were stirred at room temperature for at least 30 minutes prior to dispensation for dosing.
- Dosing formulations were prepared at the test item concentrations indicated in the table below.
- The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, protected from light. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. On each dosing day, aliquots were stirred at room temperature for at least 30 minutes prior to dispensation for dosing. The first test item dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- Study group assignments are shown in the table below.
- The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily.
- The males selected for pairing were dosed during study days 0-28 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 29 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 39-43 doses.
- Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 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 29 doses for males and 39 doses for females, these animals remained on study for a 14-day recovery period.
- The dosage 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 28-day study in rats (Haas, 2015, WIL-168233). In that study, the test item was administered to male and female rats for 28 consecutive days at dosage levels of 30, 100, 250, and 1000 mg/kg/day. There were no significant clinical signs noted at any dosage level. In addition, body weights and food consumption were unaffected by treatment. The selected route of administration for this study was oral (gavage) because this is a potential route of human exposure. Historically, this route has been used extensively for studies of this nature.

DATA ACQUISITION AND ANALYSIS
- Details of acquisition, analysis and reporting of data are attached.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The test substance in the vehicle was determined to be stable at concentrations of 5 and 230 mg/mL following 8 days of refrigerated storage in a previous study (Bystry, 2015, WIL-168232). In addition, resuspension homogeneity was assessed at concentrations of 60 to 200 mg/mL in a previous 28-day toxicity study (Haas, 2015, WIL-168233).
- Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first, fourth, fifth, sixth, seventh, and last test item dosing formulations and from the middle stratum of the first, fourth, fifth, sixth, seventh, and last control group dosing formulations. Due to difficulty with the analytical method, beginning with the fourth preparation, samples from all preparations were collected and stored refrigerated. To establish 17-day refrigerated stability, small batch sizes were prepared at the low and high concentrations used in this study. Samples were collected from the top, middle, and bottom strata of these formulations for determination of homogeneity and, following 17 days of refrigerated storage, stability. One set of samples from study weeks 0, 4, 5, and 7 was subjected to the appropriate analyses. All remaining samples were stored refrigerated (2 °C to 8 °C) as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a method validated on a previous study (Bystry, 2015, WIL-168232), as well as an alternate method that was cross-validated in this study.
Duration of treatment / exposure:
- Males: Total of 29 doses (throughout the mating period through 1 day prior to euthanasia)
- Females: Total of 39 to 43 doses (14 days prior to pairing with dosing continued until lactation Day 3)
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- Vehicle control: 15 males and 15 females
- Test item administered at 30, 100 and 250 mg/kg bw/day: 10 males and 10 females
- Test item administered at 1000 mg/kg bw/day: 15 males and 15 females
Control animals:
yes, concurrent vehicle
Details on study design:
- A flow chart summarising the study design is attached.
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 appearance, behaviour, 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. 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 labor, delayed labor) or other difficulties. Due to social housing, some observations could not be attributed to a single animal. In these instances, the observation was recorded in a separate computer protocol for the social group.

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 were presented for each interval.
- In addition, cumulative mean body weight changes were presented for the pre-mating 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, and 4. Mean gestation body weights and corresponding mean body weight changes were presented for these intervals and for the overall gestation interval (days 0-20); mean lactation body weight changes are presented for lactation days 1-4.
- Weekly body weights for females with no evidence of mating were presented in the individual report tables until delivery.
- 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
- 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 normalised 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/anima/day and g/kg/day during gestation and lactation.
- Following the breeding period, food consumption for females with no evidence of mating was measured on a weekly basis until delivery.
- 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 an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables.

FOB ASSESSMENTS
- FOB assessments were recorded for 5 animals/sex/group following approximately 28 days of dose administration (study week 4; males selected for pairing) and on lactation day 4 (females).
- The FOB used at WIL Research is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988, Moser et al., 1991; and O’Donoghue, 1989).
- 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.
- All animals were observed for the parameters as listed in the table below.
- Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer et al (1979). 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 4, males selected for pairing) or on lactation day 4 (females). Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilises 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 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.
- 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
- Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from 5 animals/sex/group at the primary necropsies (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 14-day recovery period; study day 42 for males and study day 52 for females). All males and recovery phase females were fasted overnight prior to blood collection.
- Blood for serum chemistry and haematology was collected from the retro-orbital sinus following isoflurane anaesthesia.
- 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 (clotting determinations), or no anticoagulant (serum chemistry).
- Parameters evaluated were as shown in the tables below.
Sacrifice and pathology:
MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals at the scheduled termination.
- All F0 adults were euthanised by carbon dioxide inhalation.
- Males were euthanised following completion of the mating period or following the 14-day recovery period. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded.
- Females that failed to deliver were euthanised on post-mating day 25 (females with evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
- Females not selected for pairing were euthanised following the 14-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. Clinical findings that were verified at necropsy were designated CEO.
- At the time of necropsy, the tissues and organs were placed in 10% neutral-buffered formalin (except as noted in the table below).

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.

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
- After fixation, protocol-specified tissues were trimmed according to WIL Research SOPs and the protocol.
- Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research 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 and on gross lesions from all animals in all 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. 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
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No test item-related clinical findings were noted at the detailed physical examinations or approximately 1 hour following dose administration.
- Clinical findings observed in the test item-treated groups, including hair loss and red or clear material on various body surfaces, 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 males and females in the control, 30, 100, 250, and 1000 mg/kg/day groups 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 30, 100, 250, and 1000 mg/kg/day group males were unaffected by test item administration throughout the study. A significantly (p < 0.05) lower mean body weight gain was noted for males in the 250 mg/kg/day group during study days 13 20 and significantly (p < 0.05) higher mean body weight gains were noted for males in the 100 and 1000 mg/kg/day groups for the pre-mating period (study days 0-13). However, these results did not occur in a dose-related manner and did not affect mean absolute body weights. No other differences from the control group were statistically significant.
- Females (pre-mating): Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating period (females selected for breeding) or the entire study (females selected for recovery phase). Significantly (p < 0.05) higher mean body weight gains were noted for females in the 1000 mg/kg/day group during study days 34-38 and 46-51. However, these results were transient and did not affect mean absolute body weights. No other differences from the control group were statistically significant.
- Females (gestation): Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item 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 30, 100, 250, 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):
effects observed, non-treatment-related
Description (incidence and severity):
- Males: Mean food consumption in the 30, 100, 250, 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 (pre-mating): Mean food consumption in the 30, 100, 250, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating period (females selected for breeding) or the entire study (females selected for recovery phase). None of the differences from the control group were statistically significant.
- Females (gestation): Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 30, 100, 250, and 1000 mg/kg/day groups was unaffected by test item 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 and g/kg/day, in the 30, 100, 250, 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.
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 item-related alterations in haematology and coagulation parameters at the scheduled necropsies. However, some statistically significant (p < 0.05) differences were observed when the control and test substance-treated groups were compared.
- Lower absolute basophil values were noted for females in the 100 and 1000 mg/kg/day groups at the primary necropsy. There was no dose-response relationship, values were of minimal magnitude difference, and were similar to the control group at the recovery necropsy; the differences were attributed to biological variability.
- Higher prothrombin time (PT) values were noted in the 1000 mg/kg/day group males at the recovery necropsy. Values were of minimal magnitude change and were similar to control and 1000 mg/kg/day group values at the primary necropsy; the finding was attributed to biological variability and not related to administration of test item.
- Statistically significant (p < 0.05) findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no test item-related alterations in serum chemistry parameters at the scheduled necropsies.
- However, some statistically significant (p < 0.01 or p < 0.05) differences were observed when the control and test item-treated groups were compared.
- Lower serum chloride values were noted in the 1000 mg/kg/day group males at the primary necropsy. Other serum electrolyte values were similar to the control group, the difference was of minimal magnitude, and there were no histologic correlates; the finding was attributed to biological variability.
- Higher serum urea nitrogen values were noted in the 1000 mg/kg/day group males and higher serum triglyceride values were noted in the 1000 mg/kg/day group females at the recovery necropsy. Both values were similar to the control group at the primary necropsy and were of minimal magnitude difference; the Findings were attributed to biological variability and not related to administration of test item.
- Other statistically significant differences lacked a dose-response relationship, and were attributed to biological variability.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
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. A significantly (p < 0.05) higher number of males in the 250 mg/kg/day group had slight resistance to being handled; however, this did not occur in a dose-related manner. There were no other 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).
- Neuromuscular 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).
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 item-related alterations in final body weights or organ weights at the scheduled necropsies. However, some statistically significant (p < 0.05) differences were observed when the control and test item-treated groups were compared.
- Lower absolute kidney weights were noted in the 1000 mg/kg/day group males at the recovery necropsy. Values were similar to the control group when corrected for body weight; the finding was
not considered to be related to administration of test item.
- Higher thyroid/parathyroid gland weights (absolute and relative to body and brain weights) were noted in the 1000 mg/kg/day group females at the recovery necropsy. Weights were similar between control and 1000 mg/kg/day group females at the primary necropsy, and the magnitude of change was minimal; the findings were attributed to biological variability and not related to administration of test item.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
MACROSCOPIC EXAMINATIONS
- No test item-related internal findings were observed at any dosage level in females that failed to deliver or males and females at the scheduled necropsies.
- Macroscopic findings observed in the test item-treated groups occurred infrequently and/or in a manner that was not dose-related.
- The mean numbers of unaccounted-for sites and implantation sites in the 30, 100, 250, and 1000 mg/kg/day groups were similar to the control group values.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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) or 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 analysed 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).
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no test item-related histologic changes at the primary necropsy. Histologic changes noted were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance.
- There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
no

ANALYSES OF DOSING FORMULATIONS

- Results of the analyses of dosing formulation are summarised in the tables attached.

- The analysed dosing formulations were within WIL Research SOP range for suspensions (85 % to 115 %), were homogeneous, and were stable following 17 days of refrigerated storage, with the following exceptions. The 6 mg/mL formulation prepared on 06 May 2015 was 114 % of target concentration with 12 % RSD. In addition, a peak eluting at the same time as the analyte was detected in the control group formulation prepared on 06-May-2015. The peak was believed to be caused by the initial injections of the vehicle (peanut oil), with vehicle interference carryover of the peak detected in the Group 2 formulations, forcing the higher RSD value. This was corrected by cross-validation of the method to alternate instrument settings and HPLC column. When analysed with the alternate instrumentation method, the dosing formulations were within WIL Research SOP range and no test item was detected in the analysed vehicle administered to the control group. Therefore, this did not affect the quality or integrity of the study.

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, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the 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 30, 100, 250, 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:

GUIDELINE

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. The protocol was designed in general accordance with the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (22March1996).

 

METHODS

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 30, 100, 250, 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 29 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-43 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 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 29 doses for males and 39 doses for females, these animals were assigned to a 14-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 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 (haematology and serum chemistry) were performed on 5 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 14-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, 30, 100, 250, and 1000 mg/kg/day groups survived to the scheduled necropsies. No test item-related clinical findings were noted for males or females at the detailed physical examinations or approximately 1 hour following dose administration at any dosage level.

 

Mean F0 male and female body weights, body weight gains, and food consumption in the test item groups were unaffected by test item administration throughout the study. 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 30, 100, 250, 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-related alterations in clinical pathology parameters (haematology, coagulation, and serum chemistry) were noted for F0 males and females in the 30, 100, 250, 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 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration.

 

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, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the 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 30, 100, 250, 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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Remarks on result:
other: studies conducted in accordance with OECD 422 and OECD 407
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral route

Key Study 1

A key study was performed on an analogue test item to evaluate the potential toxic effects when administered to rats for 28 days and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development. The protocol was designed in general accordance with the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (22 March 1996).

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 levelswere 30,100, 250, 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 29 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-43 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 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 29 doses for males and 39 doses for females, these animals were assigned to a 14-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 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 (haematology and serum chemistry) were performed on 5 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 14-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.

All F0 males and females in the control, 30, 100, 250, and 1000 mg/kg/day groups survived to the scheduled necropsies. No test item-related clinical findings were noted for males or females at the detailed physical examinations or approximately 1 hour following dose administration at any dosage level.

 

Mean F0 male and female body weights, body weight gains, and food consumption in the test item groups were unaffected by test item administration throughout the study. 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 30, 100, 250, 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-related alterations in clinical pathology parameters (haematology, coagulation, and serum chemistry) were noted for F0 males and females in the 30, 100, 250, 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 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration.

 

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, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the 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 30, 100, 250, 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.

Key Study 2

A key study was conducted on an analogue test item in general accordance with the OECD (2008), Test No. 407: Repeated Dose 28 Day Oral Toxicity Study in Rodents, OECD Guidelines for the Testing of Chemicals, Section 4. The objectives of the study were to evaluate the potential toxic effects of the test substance when administered via gavage to rats for 28 consecutive days, as well as to evaluate the recovery, persistence, or progression of any effects following a 14-day recovery period. This included evaluation of potential neurotoxicity by functional observational battery and motor activity assessment.

The test substance, in the vehicle (peanut oil), was administered orally by gavage once daily for 28 consecutive days to 4 groups (Groups 2-5) of Crl:CD(SD) rats. Dosage levels were 30, 100, 250, and 1000 mg/kg/day for Groups 2, 3, 4, and 5, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Groups 1 and 5 each consisted of 10 animals/sex, and Groups 2-4 each consisted of 5 animals/sex. Following 28 days of dose administration, 5 rats/sex/group were euthanized; the remaining 5 rats/sex in the control and high-dose groups were euthanized following a 14-day non-dosing (recovery) period. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (±2 days). Individual body and food weights were recorded weekly (±2 days). Functional observational battery (FOB) and locomotor activity data were recorded for 5 animals/sex/group during study week 3 and on all remaining animals during study week 5. Clinical pathology parameters (haematology, coagulation, serum chemistry, and urinalysis) were analysed for all animals scheduled for the primary (study day 28) and recovery (study day 42) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

All animals survived to the scheduled necropsy. Test substance-related clinical observations were noted during the dosing period and consisted of red material around the nose for the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females, clear material around the mouth for the 1000 mg/kg/day group males and females, and clear material around the ventral neck for the 1000 mg/kg/day group females. Recovery from these clinical observations was evident during the recovery period.

 

There were no test substance-related alterations in body weights, food consumption, clinical pathology parameters, or organ weights. In addition, there were no test substance-related macroscopic or microscopic findings noted at the scheduled necropsies. No test substance-related neurobehavioral findings (FOB or motor activity alterations) were noted at the end of the dosing and recovery periods.

Based on the results of this study, administration of test item via oral gavage to Sprague Dawley rats for 28 consecutive days was well tolerated at dosage levels of 30, 100, 250, and 1000 mg/kg/day and resulted in non-adverse clinical observations in the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day (the highest dosage level tested).

Dermal route

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. However, no evidence of systemic toxicity was demonstrated during an acute study via the dermal route involving an analogue substance and, in order to minimise the number of vertebrate animals tested, oral dosing by gavage using the same analogue was considered most appropriate for investigation of repeated dose toxicity.

Inhalation route

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. The registered substance is a low melting point solid (29 to 58 °C)with high onset boiling point (decomposition from approximately 150 °C at 101 kPa) and the vapour pressure of a close analogue was shown to be 6.27 x 10E-03 Pa at 25 °C. It is therefore expected that inhalation exposure will be low under general use conditions at ambient temperature. Lack of systemic toxicity when the registered substance or a close analogue is administered via the oral and dermal routes suggests that determined vapour pressures of 1.51 x 10E-02 Pa at 55 °C and 7.24 x 10E-02 Pa at 85 °C for the analogue substance also give no cause for concern. The inhalation route is therefore not the most applicable method of investigating repeated dose toxicity.

Justification for classification or non-classification

No treatment-related effects were reported at dose levels of 30, 100, 250 and 1000 mg/kg bw following administration of an analogue test item in two studies. Classification for Specific Target Organ Toxicity is therefore not required under the terms of Regulation (EC) No 1272/2008 and subsequent amendments.