Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 October 2016 to 25 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

various deviations with no impact on study results or integrity (see Appendix 1, attached)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

EC Number:

800-172-4

Cas Number:

398141-87-2

Molecular formula:

Complex UVCB substance

IUPAC Name:

Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Test material form:

liquid

Details on test material:

- Physical description: clear yellow liquid
- Storage conditions: room temperature protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system 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 recognised as appropriate for repeated-dose 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 (up to 15 animals/sex/group) was the minimum required to yield scientifically meaningful results.
- Crl:CD(SD) rats (77 males and 77 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 04 Oct 2016. The animals were approximately 37 days old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed 2 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. During acclimation, each animal was observed twice daily for mortality and changes in general appearance or behaviour.
- Data collection during acclimation began on 06 Oct 2016. Individual body weights and cage food weights were recorded and detailed physical examinations were performed periodically during acclimation. Ophthalmic examination data was also recorded for animals 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 Andersons, Cob Products Division, Maumee, OH).
- Animals whose cage mate(s) were removed from the study (morbidity or unscheduled death) were not re-paired and remained individually housed for the remainder of the study.
- The cages were cleaned and changed routinely at a frequency consistent with maintaining good animal health. 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 objectives of this study. The results of these analyses are maintained at Charles River.
- The animals were temporarily separated as necessary to allow for the performance of protocol-specified activities (see Appendix 1 - Study Protocol and Deviations, attached). Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitised weekly.

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 Charles River. 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 when food, but not water, was withheld.
- Municipal water supplying the facility was analysed for contaminants according to SOPs. The results of the diet and water analyses are maintained at Charles River. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

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 73 °F ± 5 °F (23 °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 72.4 °F to 73.7 °F (22.4 °C to 23.2 °C) and mean daily relative humidity ranged from 34.8% to 64.1% during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Lot numbers 2FA0334 (expiry date 31 January 2017) and 1FE0684 (expiry date 31 May 2017)

Details on oral exposure:

PREPARATION
- The vehicle solution was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature (18 °C to 24 °C), protected from light. The vehicle was mixed throughout the preparation, sampling, and dose administration procedures. 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 at room temperature (18 °C to 24 °C), protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- On 10 Oct 2016 (8 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 computerised 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 treatment groups and housed in social 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 Charles River rat colony.
- The control and 500 mg/kg/day groups (Groups 1 and 6, respectively) each consisted of 15 males and 15 females, and the 5, 25, 125, and 250 mg/kg/day groups (Groups 2, 3, 4, and 5, respectively) each consisted of 10 males and 10 females. The animals were approximately 7 weeks old at the initiation of dose administration. Individual body weights ranged from 146 g to 190 g for males and from 118 g to 169 g for females at randomization, and from 208 g to 282 g for males and from 151 g to 208 g for females at the initiation of dose administration (see Appendix 1 - Study Protocol and Deviations, attached).

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test substance formulations were administered orally by gavage via a syringe equipped with an Instech feeding tube (Instech Solomon, Plymouth Meeting, PA) once daily for 90/91 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.
- The first day of dosing was Study Day 0; the first week of dosing was Study Week 0.
- Study group assignment is shown in the table below.
- The dosage levels were determined from results of a previous 28-day oral gavage study4 in rats with this test substance in which the no-observed-adverse-effect level (NOAEL) was determined to be 500 mg/kg/day. It was anticipated that the high-dosage level would show test substance-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. Though priority was given to detecting a dose-related trend, it was expected that the low-dosage level would be a no-observed-effect-level (NOEL).
- The selected route of administration for this study was oral (gavage) because this is a potential route of exposure in humans. Historically, this route has been used extensively for studies of this nature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING AND ANALYSIS
- The test substance has been determined to be stable and homogeneous over the range of concentrations used on this study for at least 8 days at room temperature (18 °C to 24 °C). Therefore, further evaluation of stability was not conducted as part of the current study.
- Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 1 and 100 mg/mL dosing formulations and from the middle of the control and 5, 25, and 50 mg/mL dosing formulations.
- For resuspension homogeneity determinations, a volume of the 1 and 100 mg/mL formulations similar in size to the amount needed for 1 day was stored at room temperature (18 °C to 24 °C), protected from light, for 8 days. After remixing these formulations for a minimum of 30 minutes using a magnetic stirrer, samples were collected from the top and bottom of the formulations and analysed.
- Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared for Study Weeks 0, 3, 7, and 12. One duplicate set was analysed and the remaining duplicate set was stored at room temperature (18 °C to 24 °C) and retained as backup samples.
- All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method using with charged aerosol detection.

Duration of treatment / exposure:

90 or 91 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Doses / concentrations 5

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 6

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

- Group 1 (control): 15 animlas/sex
- Groups 2, 3, 4 and 5 (5, 25, 125 and 250 mg/kg/day): 10 animals/sex
- Group 6 (500 mg/kg/day): 15 animals/sex

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.
- Animals found dead were examined macroscopically as soon as possible to ensure that tissues were not lost due to autolysis.

CLINICAL OBSERVATIONS
- Clinical examinations were performed at the time of dose administration and 1 to 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 within 4 days of receipt, on the day of randomisation, weekly (± 2 days) during the study period, and on the days of the scheduled necropsies.
- Daily observations during the recovery period were not necessary on days when detailed physical examinations were conducted.
- In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal; only positive findings were recorded.
- Any observations noted outside of the above-specified postdosing intervals were also recorded.
- In addition, a separate computer protocol was used to record any observations not associated with the postdosing intervals and treatments.

BODY WEIGHTS
- Individual body weights were recorded within 4 days of receipt, on the day of randomization,
Study Day 0 (prior to dosing), weekly (± 2 days) during the study period, and on the day prior to
the first day of the scheduled necropsies (nonfasted).
- The second weekly body weights collected during Study Week -2 are identified in the text and on the report tables as Study Week -1.
- The last nonfasted body weights were collected on the second to last day of Study Week 12 or 16, which was the day prior to each animal’s scheduled necropsy. These second weekly body weights during Study Week 12 or 16 are identified in the text and on the report tables as Study Week 13 or 17, respectively.
- Mean body weights and mean body weight changes were calculated for the corresponding intervals.
- Final body weights (fasted) were recorded on the day of the scheduled necropsies.

FOOD CONSUMPTION
- Cage food weights were recorded weekly (± 2 days) beginning following randomisation and throughout the study period. Food consumption was calculated as g/animal/day for the corresponding body weight intervals.
- The last food weights were collected on the second to last day of Study Week 12 or 16, which was the day prior to each animal’s scheduled necropsy. These second weekly food consumptions during Study Week 12 or 16 are identified in the text and on the report tables as Study Week 13 or 17, respectively.
- When food consumption could not be measured for a given interval (due to spillage, termination of the social group, weighing error, obvious erroneous value, selection for necropsy, etc.), the appropriate interval was footnoted as "NA" on the individual tables.

FOB ASSESSMENTS
FOB assessments were recorded for all animals during Study Week 12. The FOB utiliSed at Charles River is based on previously developed protocols.
- Testing was performed by the same biologists, whenever 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 following parameters as described in the table below.
- Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer et al. 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 all surviving animals during Study Week 12.
- Motor activity, recorded after the completion of the FOB, assessment was conducted 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 and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected from all animals assigned to the scheduled necropsies (Study Weeks 12/13 and 16/17).
- The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected for haematology and serum chemistry evaluation via the jugular vein. - Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals euthanised by inhalation of carbon dioxide.
- Blood was collected into tubes containing potassium (K2) EDTA (haematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).
- The parameters listed in the tables below were evaluated.

OPTHALMIC EXAMINATIONS
- Ocular examinations were conducted on all animals during acclimation (07 Oct 2016; Study Week -2) and near the end of the dosing period (13 Jan 2017; Study Week 12).
- All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

Sacrifice and pathology:

MACROSCOPIC EXAMINATIONS
- A complete necropsy was conducted on all animals found dead or at the scheduled necropsies.
- Animals were euthanised by carbon dioxide inhalation followed 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.
- Clinical findings that were confirmed macroscopically were designated CEO on the individual macroscopic data tables.
- The tissues and organs listed in the table below were collected from all animals and placed in 10% neutral-buffered formalin (except as noted).

ORGAN WEIGHTS
- Organs listed in the table below were weighed from all animals at the scheduled necropsies.
- Paired organs were weighed together. Organ to final body weight and organ to brain weight ratios were calculated. When organ weights could not be determined for an animal (due to weighing error, lost or damaged organ, etc.), group mean values were calculated using the available data (see Appendix 1 - Study Protocol and Deviations, attached).

HISTOLOGY AND MICROSCOPIC EXAMINATION
- After fixation, protocol-specified tissues were trimmed according to Charles River SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to Charles River SOPs, mounted on glass microscope slides, and stained with haematoxylin and eosin.
- Microscopic examination was performed on all tissues listed from all animals found dead and in the control and 500 mg/kg/day groups at the primary necropsy.
- Gross lesions were prepared from all animals in the 5, 25, 125, and 250 mg/kg/day groups (Groups 2–5) euthanised at the primary necropsy and from all animals euthanised at the recovery necropsy (see Appendix 1 - Study Protocol and Deviations, attached).
- After sectioning for routine pathology, kidney blocks (from all groups at all unscheduled and scheduled necropsies) were shipped to PAI Durham for immunohistochemical (IHC) staining. IHC staining of the kidneys from animals found dead and from animals in the control and 500 mg/kg/day groups euthanised at the primary necropsy were conducted. In addition, immunohistochemically-stained kidney sections were examined from all males euthanised at the primary and recovery necropsies and from all females in the control and 500 mg/kg/day group at the primary necropsy in an attempt to determine if any renal histopathological changes noted were mediated by alpha-2u globulin nephropathy. The kidneys (males only), liver, and thyroids were identified as target tissues and were examined from all animals in the 5, 25, 125, and 250 mg/kg/day groups at the primary and recovery necropsies.
- Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate (see Appendix 1 - Study Protocol and Deviations, attached).
- 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.
- A formal histopathology peer review was not conducted as part of this study.

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 in the 125, 250, and 500 mg/kg/day group males and/or females.
- A test substance-related clinical observation of clear material around the mouth was noted in the 125 mg/kg/day group males and the 250 and 500 mg/kg/day group males and females.
- Salivation, yellow material around the mouth, and yellow material around the urogenital area were noted in the 500 mg/kg/day group males and females. In addition, yellow material around the urogenital and/or anogenital areas was noted in the 250 mg/kg/day females.
- There were no test substance-related clinical observations noted during the recovery period.
- All other clinical observations 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:

mortality observed, non-treatment-related

Description (incidence):

- Two animals died prior to the scheduled necropsy: one 125 mg/kg/day group male (No. 8381) found dead on Study Day 88 and one 25 mg/kg/day group female (No. 8487) found dead on Study Day 82. There were no clinical observations noted for either animal. Test item-related higher body weights and cumulative body gains were noted for both animals. Neither animal had test item-related macroscopic or microscopic findings considered to be the cause of death.
- No macroscopic observations were noted in the 125 mg/kg/day group male. Microscopic findings consisted of minimal accumulation of hyaline droplets within renal proximal convoluted tubules, chronic interstitial nephritis, pulmonary perivascular mononuclear infiltrates, and mild thymic haemorrhage. Sections of kidney immunohistochemically stained for alpha-2u globulin demonstrated a positive staining pattern with minimally increased globular staining when compared to the control group males. The cause of death was undetermined.
- Macroscopic findings in the 25 mg/kg/day group female consisted of edematous thymus which correlated microscopically with oedema and ventral abdominal skin mass described as firm and ruptured, which correlated with mammary gland adenoma with necrosis, thrombosis, and mineralisation. Other microscopic findings were consistent with response to stress, including lymphoid depletion in the thymus, spleen, and lymph nodes and adrenal cortical hypertrophy. Centrilobular coagulative necrosis was noted in the liver; the finding was consistent with ischemia and may have been related to necrosis and thrombosis within the mammary gland adenoma. For this reason, the cause of death was considered likely to have been related to complications associated with the adenoma. Spontaneous mammary gland adenomas are uncommon in this age group of rats. The finding was noted in a single lower dose group animal and no proliferative mammary gland changes were noted in the 500 mg/kg/day groups; the finding thus was attributed to spontaneous change and not related to administration of test item. Immunohistochemically stained sections of kidney were negative for alpha-2u globulin, similar to the control group females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Test substance-related effects on body weights were noted in the 250 and 500 mg/kg/day group females.
- Slightly higher mean cumulative body weight gains were noted beginning during Study Week 1 in the 250 and 500 mg/kg/day group females, which continued until the primary necropsy (Study Week 13). During Study Week 13 (the day of the primary necropsy for the 250 and 500 mg/kg/day group females), the mean body weights in the 250 and 500 mg/kg/day females were 2.3% and 3.2% higher, respectively, than the mean control group body weight. In addition, body weight gains in the 500 mg/kg/day females were comparable to control group values during the recovery period.
- There were no other test substance-related effects on body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Test substance-related effects on food consumption were noted in the 250 and 500 mg/kg/day group females.
- During the dosing period, higher mean food consumption (occasionally statistically significant) compared to the control group was noted in the 250 and 500 mg/kg/day group females starting during Study Weeks 1 to 2 and throughout the study. The slight increase in food consumption correlated with the slightly higher cumulative mean body weight gains noted in the 250 and 500 mg/kg/day group females. During the recovery period, slightly higher food consumption values were still noted in the 500 mg/kg/day group females.
- There were no other test substance-related effects on food consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups.
- All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- There were no test item-related alterations in haematology. Potential test item-related changes in coagulation consisted of prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) values.
- Prolonged PT and APTT were noted in the 500 mg/kg/day group males at Study Week 12/13. Minimally to mildly prolonged PT was noted primarily in Male Nos. 8362 and 8376 and minimally to mildly prolonged APTT was noted primarily in Male No. 8376. These changes were not noted at Study Week 16/17, indicating recovery. The underlying mechanism of prolonged PT and APTT was not evident in this study. These changes were considered to be potentially related to test material administration based on magnitude of change; however, they lacked correlation with related changes in haematology and serum chemistry.
- Shortened PT was noted in the 500 mg/kg/day group females at Study Weeks 12/13 and 16/17. Shortened PT was not considered to be test item-related because it was in a direction of no known interpretative significance.
- Higher absolute neutrophil and monocyte counts were noted in the 500 mg/kg/day group males at Study Week 16/17. These changes were of minimal magnitude and not considered to be related to administration of test material because similar alterations were not noted during the primary phase of this study.
- Remaining differences in haematology and coagulation parameters, including those that may have been statistically significant, were not considered test item-related and were attributed to biologic variation because there were no correlations with other related clinical pathology changes, no dose-response, and/or the change was of a magnitude commonly observed in Sprague Dawley rats under similar study conditions.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related alterations in serum chemistry parameters.
- Statistically significant higher phosphorus was noted in the 250 and 500 mg/kg/day group females at Study Week 12/13. Minimally higher phosphorus was noted in 250 mg/kg/day group Female Nos.8450 and 8472 and 500 mg/kg/day group Female Nos. 8449, 8453, and 8461. Higher phosphorus was not considered to be related to test material administration based on low incidence and low magnitude of change.
- Similarly, minimally higher alanine aminotransferase (ALT) was noted in a single animal (Male No. 8362) in the 500 mg/kg/day group at Study Week 12/13. This change was unlikely related to test item administration based on low incidence and low magnitude of change.
- A moderately higher triglyceride concentration was noted in Male No. 8436 in the 500 mg/kg/day group at Study Week 16/17. This change was not considered to be related to test material administration because similar alteration was not noted during the primary phase of the study.
- Remaining differences in serum chemistry parameters, including those that may have been statistically significant, were not considered test item-related and were attributed to biologic variation because there were no correlations with other related clinical pathology changes, no dose response, and/or the change was of a magnitude commonly observed in Sprague Dawley rats under similar study conditions.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related alterations in urinalysis parameters.
- Lower urine pH was noted in the 125, 250, and 500 mg/kg/day group males at Study Week 12/13. This change was unlikely related to test item administration because most values were within the range of control animals and there were no concurrent changes in serum chemistry (i.e. changes in electrolytes).
- Remaining differences in urinalysis parameters, including those that may have been statistically significant, were not considered OS43451AU-related and were attributed to biologic variation because there were no correlations with other related clinical pathology changes, no dose response, and/or the change was of a magnitude commonly observed in Sprague Dawley rats under similar study conditions.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Home cage observations: Home cage 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 12 evaluation.
- 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 12 evaluation.
- 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 12 evaluation.
- 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 12 evaluation.
- 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 12 evaluation.
- 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 12 evaluation.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Test item-related, statistically significantly higher mean liver, thyroid/parathyroid gland, and kidney weights were noted in the 125, 250, and/or 500 mg/kg/day groups at the primary necropsy.
- Higher kidney weights were noted in the 125 (males), 250, and 500 mg/kg/day groups with statistical significance in all but the 250 mg/kg/day group males (absolute) and the 250 and 500 mg/kg/day group females (relative to brain weight). No microscopic correlates were noted and a definitive dose-response relationship was lacking.
- Higher mean liver weights were noted in the 250 (relative to final body weight) and 500 (absolute and relative to final body and brain weights) mg/kg/day group males, and in the 125 (relative to final body weight), 250, and 500 (absolute and relative to final body and brain weights) mg/kg/day group females. Higher weights correlated microscopically with hepatocellular hypertrophy and were considered to be an adaptive response to test material administration.
- Higher mean thyroid/parathyroid gland weights (absolute and relative to final body and brain weights) were noted in the 500 mg/kg/day group males and females, with statistical significance in the males. Higher weights correlated with follicular cell hypertrophy, and were considered to be an adaptive response.
- There were no other test substance-related effects on organ weights. However, 1 statistically significant difference was observed when the control and test substance-treated groups were compared. Higher mean epididymides weight (relative to final body weights) was noted in the 500 mg/kg/day group males. There were no microscopic correlates, other male reproductive organ weights were similar to the control group, and weights were within the historical control database range; therefore, the finding was not considered to be related to administration of test item.
- At recovery, higher mean kidney (absolute), liver (absolute and relative to final body weight), and thyroid/parathyroid gland (relative to final body weight) weights persisted in the 500 mg/kg/day group females, with no microscopic correlates. Mean kidney, liver, and thyroid/parathyroid gland weights in the 500 mg/kg/day group males were similar to the control group, consistent with recovery.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Review of the gross necropsy observations revealed no findings that were considered to be directly associated with administration of test item.
- One finding deserves mention, however. Mottled kidneys were observed in a 250 mg/kg/day group male (No. 8389) at the primary necropsy, which correlated microscopically with mild chronic progressive nephropathy. Chronic progressive nephropathy is a common spontaneous finding in this age group of male rats; however, microscopic features of alpha-2u globulin nephropathy were also noted (hyaline droplet accumulation, single cell necrosis, and medullary granular casts), suggesting that the finding may have been related to test material administration via increased alpha-2u accumulation.

Neuropathological findings:

no effects observed

Description (incidence and severity):

MOTOR ACTIVITY
- Motor activity patterns (total and ambulatory activity counts) were unaffected by test substance
administration. T
- here were no statistically significant changes for the test substance-treated males and females when compared to the control group at the Study Week 12 evaluation.
- 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.
- No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated on Study Week 12.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Test item-related microscopic findings were noted in the kidney of the 5, 25, 125, 250, and 500 mg/kg/day group males; liver of the 125 (females), 250, and 500 mg/kg/day groups; and thyroid glands of the 500 mg/kg/day groups at the primary necropsy.
- Test material-related findings at the primary necropsy are summarised in the attached table.
- Renal findings noted in the 125, 250, and/or 500 mg/kg/day group males consisted of increased alpha-2u globulin-immunopositive hyaline droplet accumulation, single cell necrosis within proximal convoluted tubule (PCT) epithelial cells, granular casts, and a higher incidence and/or severity of chronic progressive nephropathy (CPN); hyaline droplet accumulation was additionally noted in the 5 and 25 mg/kg/day group males. Hyaline droplet accumulation in test item-treated males was characterised by increased brightly eosinophilic, variably sized and shaped intracytoplasmic droplets within PCT epithelium. Immunohistochemically, the normal pattern of punctate immunopositivity with scattered globular accumulations noted in the control group males was replaced by increased intracellular globular immunopositivity in irregular linear cortical tubular arrays. Single cell necrosis was characterised by rare rounded, hypereosinophilic necrotic tubular epithelial cells within PCTs. Granular casts were characterised by rare to few dilated tubules containing cellular debris at the junction of the inner and outer stripe of the outer medulla. CPN was characterised by a spectrum of tubular and interstitial changes consisting of basophilic tubules, thickened basement membranes, and mononuclear cell infiltrates. There were no clinical pathology correlates. The spectrum of renal findings noted in the 125, 250, and 500 mg/kg/day group males was consistent with alpha-2u globulin nephropathy.
- Hepatocellular hypertrophy was noted in the 125 (female), 250, and 500 mg/kg/day groups and was characterised by enlarged hepatocytes with increased eosinophilic cytoplasm in a primarily centrilobular distribution. The finding correlated with higher mean liver weights. Thyroid follicular cell hypertrophy was noted in the 500 mg/kg/day group males and females and was characterised by follicles lined by cuboidal to low columnar follicular cells with variable cytoplasmic vacuolation. The finding correlated with higher thyroid/parathyroid gland weights.
- There were no other test material-related histologic changes at the primary necropsy. Remaining 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.
- At recovery, renal findings noted in the 500 mg/kg/day group males consisted of increased alpha-2u globulin immunopositivity, granular casts, and higher incidence/severity of CPN. Hepatocellular and thyroid follicular cell hypertrophy were not noted in the 500 mg/kg/day group males and females.
- Test item-related related findings at the recovery necropsy are summarised in the attached table.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects relevant to humans at highest dose level tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

ANALYSIS OF DOSING FORMULATIONS

-The analysed dosing formulations contained 89.7% to 112% of the test substance which was within the Charles River SOP range of target concentrations for suspensions (85% to 115%), and were homogeneous.

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

- Results of homogeneity and concentration analyses are attached.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, oral administration of test item to Crl:CD(SD) rats at dosage levels of 5, 25, 125, 250, and 500 mg/kg/day for a minimum of 90 days was generally well tolerated at all dosages. Adverse microscopic renal changes related to a well-known male rat-specific alpha-2u globulin accumulation were noted in male rats at ≥ 125 mg/kg/day. All other findings were considered to be nonadverse. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 25 mg/kg/day for male rats and 500 mg/kg/day for female rats tested in this study. However, because the renal changes observed in the male rats in this study were in association with alpha-2u globulin accumulation, which is a well-known male rat-specific effect, not occurring in female rats or other species, including humans, and thus are of no human relevance, this effect should be excluded from any subsequent human risk assessment. Thus, in the absence of the alpha 2u globulin accumulation effect, the NOAEL relevant for human risk assessment is considered to be 500 mg/kg/day, the highest dosage level tested for both sexes of rats in this study.

Executive summary:

GUIDELINE

The objectives of this study were to evaluate the potential toxicity of the test item when administered daily by oral gavage to Sprague Dawley rats for a minimum of 90 consecutive days, as well as to evaluate the recovery, persistence, or progression of any effects following a minimum of a 28-day recovery period. The protocol was designed in general accordance with OECD Guideline for the testing of Chemicals 408 (September 1998).

 

METHODS

Test item in the vehicle (corn oil) was administered orally by gavage once daily for a minimum of 90 consecutive days to 5 toxicology groups (Groups 2, 3, 4, 5, and 6) of Crl:CD(SD) rats. Dosage levels were 5, 25, 125, 250, and 500 mg/kg/day for Groups 2, 3, 4, 5, and 6, 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 6 each consisted of 15 animals/sex and Groups 2–5 each consisted of 10 animals/sex. Following a minimum of 90 consecutive days of dose administration, 9–10 animals/sex/group were euthanised; the remaining 5 animals/sex in the control and high-dose groups were euthanised following a 28/29-day nondosing (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 weights and cage food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and motor activity data were recorded for all animals during Study Week 12. Ophthalmic examinations were performed during Study Weeks -2 and 12. Clinical pathology parameters (haematology, coagulation, serum chemistry, and urinalysis) were analysed for all animals assigned to the primary (Study Week 12/13) and recovery (Study Week 16/17) 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

One female (No. 8487) in the 25 mg/kg/day group was found dead prior to dosing on Study Day 82. This animal was noted with a ruptured mass at the gross necropsy examination and was considered to be the likely cause of death.

 

One male (No. 8381) in the 125 mg/kg/day group was found dead prior to dosing on Study Day 88. There were no clinical observations noted and no macroscopic findings noted at the gross necropsy. The cause of death was undetermined.

 

All other animals survived to the scheduled necropsy. There were no test substance-related effects on haematology, serum chemistry, urinalysis, functional observational battery parameters, or motor activity. There were no test substance-related ophthalmic or macroscopic findings. Higher prothrombin time and activated partial thromboplastin time noted in the 500 mg/kg/day group males at Study Week 12/13 were considered to be potentially related to test material administration.

 

Test substance-related clinical observations noted in the 250 and 500 mg/kg group males and/or females were clear and/or yellow material around the mouth. Salivation, yellow material around the mouth, and yellow material around the urogenital and/or anogenital areas were noted in the 250 mg/kg/day group females and/or the 500 mg/kg/day group males and/or females. In addition, clear material around the mouth was noted in the 125 mg/kg/day group males. There were no clinical observations noted during the recovery period.

 

Test substance-related, nonadverse higher body weights and correlating higher food consumption were noted for the 250 and 500 mg/kg/day group females throughout the dosing period. The effects on body weights and food consumption did not persist during the recovery period for the 250 mg/kg/day group females. Test substance-related higher food consumption persisted during the recovery period for the 500 mg/kg/day group females.

 

Test substance-related changes in organ weights were noted in the 125, 250, and/or 500 mg/kg/day group males and females at the primary necropsy. Higher kidney weights were noted in the 125 mg/kg/day group males and the 250 and 500 mg/kg/day group males and females. There were no microscopic correlates or a definitive dose-response noted. Higher mean liver weights were noted in the 125 mg/kg/day group females and the 250 and 500 mg/kg/day group males and females. Higher weights correlated microscopically with hepatocellular hypertrophy and were considered to be an adaptive response to test item administration. Higher mean thyroid/parathyroid gland weights were noted in the 500 mg/kg/day group males and females, correlated with follicular cell hypertrophy, and were considered to be an adaptive response. At the recovery necropsy, higher mean kidney, liver, and thyroid/parathyroid gland weights persisted in the 500 mg/kg/day group females, with no microscopic correlates. Kidney, liver, and thyroid/parathyroid gland weights in the 500 mg/kg/day group males were similar to the control group, consistent with recovery.

 

At the primary necropsy, test item-related microscopic findings noted in the kidneys of the 125, 250, and/or 500 mg/kg/day group males consisted of increased alpha-2u globulin-immunopositive hyaline droplet accumulation, single cell necrosis within proximal convoluted tubule (PCT) epithelial cells, granular casts, and a higher incidence and/or severity of chronic progressive nephropathy (CPN); hyaline droplet accumulation was additionally noted in the 5 and 25 mg/kg/day group males. Hepatocellular hypertrophy was noted in the liver of the 125 mg/kg/day females and the 250 and 500 mg/kg/day group males and females and correlated with higher liver weights. Thyroid follicular cell hypertrophy was noted in the 500 mg/kg/day group males and females and correlated with higher thyroid/parathyroid gland weights. At recovery, renal findings noted in the 500 mg/kg/day group males consisted of increased alpha-2u globulin immunopositivity, granular casts, and higher incidence/severity of CPN. Hepatocellular and thyroid follicular cell hypertrophy were not noted in the 500 mg/kg/day group males and females at the recovery necropsy.

 

CONCLUSION

Based on the results of this study, oral administration of test item to Crl:CD(SD) rats at dosage levels of 5, 25, 125, 250, and 500 mg/kg/day for a minimum of 90 days was generally well tolerated at all dosages. Adverse microscopic renal changes related to a well-known male rat-specific alpha-2u globulin accumulation were noted in male rats at ≥ 125 mg/kg/day. All other findings were considered to be nonadverse. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 25 mg/kg/day for male rats and 500 mg/kg/day for female rats tested in this study. However, because the renal changes observed in the male rats in this study were in association with alpha-2u globulin accumulation, which is a well-known male rat-specific effect, not occurring in female rats or other species, including humans, and thus are of no human relevance, this effect should be excluded from any subsequent human risk assessment. Thus, in the absence of the alpha 2u globulin accumulation effect, the NOAEL relevant for human risk assessment is considered to be 500 mg/kg/day, the highest dosage level tested for both sexes of rats in this study.