Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Appearance/physical state: Clear, light brown, viscous liquid
- Storage conditions: 18 °C to 24 °C protected from light

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
- 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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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).