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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 28, 2014 to December 15, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & UP EPA test guidelines in compliance with GLP.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
This study was conducted in accordance with the protocol except for the following unplanned protocol deviations. The following events occurred as the result of an unintended deviation from the protocol.

At arrival, several of the male animals weighed less than the protocol-specified minimum weight of 130 g. This range was provided in order to ensure that animals of similar weight, age, and condition were used for study. Despite this deviation, these animals were adequately similar to accomplish the objectives of the study.

During Week -1, grip strength was not recorded for one male at 100 mg/kg/day (animal number 3010) during the neurobehavioral examinations.

Animals were single-housed for FOB evaluations, rather than 2 to 3 animals per cage.

At the terminal urinalysis, the following animals had urine samples of insufficient volume to analyze all parameters: one female at 0 mg/kg/day (animal number 1503), one female at 100 mg/kg/day (animal number 3503), and one male and one female at 325 mg/kg/day (animal numbers 4004 and 4508, respectively). Parameters reported are urine volume, color, clarity, specific gravity, and microscopy of spun deposit.

At the recovery urinalysis, the following animals had urine samples of insufficient volume to analyze all parameters: one female at 0 mg/kg/day (animal number 1511) and one female at 325 mg/kg/day (animal number 4512). Parameters reported are urine volume, color, clarity, specific gravity, and microscopy of spun deposit.

On Day 79, the humidity of the animal room was outside the protocol-specified range. This was a physiologically insignificant and/or transient environmental
deviation, and as such, did not impact the homeostasis of the Test System.

During the study, posture, reactivity to handling, and the home cage activity were not recorded during the detailed clinical observations. The whole animal was observed for signs of abnormalities in the home cage and any observation present was recorded. This minor documentation error will have no impact on study.
In the opinion of the Study Director, these deviations did not affect the quality or integrity of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear colorless liquid
Details on test material:
Date Received: November 20, 2013
Supplier: Chemtura Manufacturing Ltd
Manchester, Great Britain
Amount Received: Approximately 2,116.1334 mL
Label Identification: Reofos 35
Batch Number: 2012123125
Physical Characteristics: Clear colorless liquid
Retest Date: March 22, 2016
Storage: Room temperature
TMC Number: 130B9E

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
A total of 60 male and 60 female experimentally naïve CD® [Crl:CD®(SD)] rats (approximately 6 weeks of age at receipt), were received from Charles River Laboratories, Portage, Michigan, on January 20, 2014. During the 8-day acclimation period, the animals were observed daily with respect to general health and any signs of disease. All animals were weighed on the day of receipt and given a detailed clinical examination prior to selection for study.

Animals assigned to study had body weights within ±20% of the mean body weight for each sex. Extra animals obtained for the study, but not placed on study, were euthanized via carbon dioxide inhalation. Euthanasia was confirmed via pneumothorax puncture and the carcasses were discarded.
Each animal was assigned an animal number to be used in the Provantis™ data collection system and was implanted with a microchip bearing a unique identification number. The individual animal number, implant number and study number comprised a unique identification for each animal. Each cage was identified by the animal number, study number, group number, and sex.

The animals were housed in pairs (same sex pairs) in solid bottom cages with nonaromatic bedding in an environmentally controlled room. Due to an odd number of animals per group, one Group 1 and 4 animal/sex was housed with a source animal to maintain social housing requirements. The animals were individually housed during times of urine collection for clinical pathology analysis. Animal enrichment was provided according to MPI Research SOP. The animals remained within the testing environment to reduce the potential influence of environmental stressors associated with cage changes and/or movement during the testing period. Fluorescent lighting was provided for approximately 12 hours per day. The dark cycle was interrupted intermittently due to study-related activities. Temperature and humidity were continuously monitored, recorded, and maintained to the maximum extent possible within the ranges of 66 to 77°F and 30 to 70%, respectively.

Block Lab Diet® (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum, except during designated periods. Tap water was available ad libitum via an automatic watering system. The Study Director is not aware of any potential contaminants likely to be present in the diet or water that would have interfered with the results of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Fresh vehicle, corn oil, was dispensed for use on study weekly and was stored refrigerated (2 to 8°C) in amber glass bottles.
The test article, Reofos 35, was used as received from the Sponsor. No adjustment was made for purity when preparing the test article formulations. Formulations of the test article were prepared weekly by diluting with an appropriate amount of the vehicle at the desired concentrations of 5, 20, and 65 mg/mL, stirred using a magnetic stir bar and stir plate, and stored refrigerated (2 to 8°C) in amber glass bottles until picked up for dosing.

The vehicle and test article were administered once per day for 91 consecutive days during the study via oral gavage. The dose levels were 25, 100, and 325 mg/kg/day and administered at a dose volume of 5 mL/kg. The control group received the vehicle in the same manner as the treated groups. The vehicle and test article were withdrawn from stirred formulations. Individual doses were based on the most recent body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples (see table in "Any other information") were collected while the formulations were stirring using a positive displacement pipette, and placed into 15 mL amber glass bottles.

Analyses
Samples were refrigerated (2 to 8°C) until shipped on gel packs to the MPI Research, Inc., site at State College, Pennsylvania, for analysis. All analytical work was conducted by MPI Research, Inc., using an analytical method developed by MPI Research, Inc., and validated under MPI Research Study Number 399-246. Analysis deviations are presented below.
The protocol states that samples are to be stored refrigerated (2 to 8°C). However, unused portions of dose formulation samples were not returned to refrigerated storage after analysis.
There is no suspected impact on the study, as the samples were not re-tested following ambient storage.
The protocol states that dose formulation samples are to be stored refrigerated until analysed (within 10-days of collection). Dose formulation samples were not analyzed within 10 days of collection; however, they were analyzed within the established stability interval listed in the analytical method (28 days at refrigerated conditions), so there is no suspected impact on the study.
Duration of treatment / exposure:
The vehicle and test article were administered for 91 consecutive days during the study via oral gavage.
Frequency of treatment:
The vehicle and test article were administered once per day during the study via oral gavage.
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 100, and 325 mg/kg/day and administered at a dose volume of 5 mL/kg.
Basis:
actual ingested
No. of animals per sex per dose:
50 male and 50 female animals were assigned to the control and treatment groups - group assignement reported in table for - See any other information.
Control animals:
yes, concurrent vehicle
Details on study design:
Justification for Route of Administration
The oral route is one of the potential routes of human exposure to this test article.

Justification of Dose Levels
The dose levels were selected by the Sponsor on the basis of available data from a four-week dose range-finding toxicity study (MPI Research Study Number 399-242). Decreases in mean body weight were observed (up to 15%) in males administered doses of up to 500 mg/kg/day during the last two weeks of the study. Dose levels up to 325 mg/kg/day were evaluated for 91 consecutive days. The low and mid dose are at approximate log intervals and were chosen to provide a graded-response.

5 animals from the control and high dose groups remained on study for a designated 28-day recovery period and were necropsied on Day 120.
Positive control:
Postive control not utilised in this study

Examinations

Observations and examinations performed and frequency:
Cage side Observations: All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily.

Cage side Clinical Observations: A cage side clinical examination of each animal was performed once daily during the study. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, and nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior. If warranted the animal was removed from the cage for closer inspection.

Neurobehavioral Examinations: A neurobehavioral examination of each animal was performed once prior to the initiation of test article administration and weekly during the study. Observations were made outside the animal’s home cage in a standard arena using an applicable scoring system at approximately the same time and day (morning), each week. Observations were carefully recorded, and an effort was made to ensure that variations in the test conditions were minimal. The observations included, but were not limited to changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded. Evidence of straub tail was also recorded if present. Examinations are not required during the week of FOB evaluation.

Functional Observational Battery Observations: Functional Observational Battery (FOB) evaluations were conducted without knowledge on the part of the testers of the treatment groups on all animals during Week 13 of test article administration (1 hour postdose) and during recovery. FOB evaluations included those conducted in the home-cage, during handling, in the open-field, and others. During open-field evaluations, each animal was placed in a black plexiglass™ box and observed for a minimum of 3 minutes. The parameters evaluated in the FOB were based on those outlined in Moser, et al. The observations included, but were not limited to, evaluation of activity and arousal, posture, rearing, bizarre behavior, clonic and tonic movements, gait, mobility, stereotypy, righting reflex, response to stimulus (approach, click, tail pinch, and touch), palpebral closure, pupil response, piloerection, exophthalmus, lacrimation, salivation, and respiration. Qualitative and/or quantitative measures of defecation and urination were also recorded. Forelimb and hindlimb grip strength was measured using the procedure described by Meyer, et al., and hindlimb splay was quantitatively measured as described by Edwards and Parker. Pain perception was assessed by measuring the latency of response to a nociceptive (thermal) stimulus when each animal was placed on a hot plate apparatus set to 52°C (± 1°C) as described by Ankier. Body weight and temperature were also measured..

Locomotor Activity: Locomotor activity evaluations were conducted on all animals from 1 to 3 hours postdose during Week 13 of test article administration and during recovery. Each animal was placed into the assigned Hamilton-Kinder enclosure for monitoring. The duration of monitoring was 60 minutes with the data summarized into 10 minute segments. A range of different activities were assessed in a three dimensional array and were recorded. Only basic movement, fine movement, rearing, and distance (cm) were used in comparisons between treated and control animals as the most representative activity parameters.

Body Weights: Body weights for all animals were measured and recorded weekly during the study, beginning on Week -1.

Food Consumption: Food consumption was measured and recorded weekly during the study.

Ophthalmoscopic Examinations: Ophthalmoscopic examinations were conducted on all animals pretest and all animals again prior to the terminal and recovery necropsies.

Clinical Pathology: Clinical pathology evaluations were conducted on designated animals prior to the terminal and recovery necropsies. The animals had access to drinking water but were fasted overnight prior to scheduled sample collection. Blood samples (approximately 4 mL) were collected from the vena cava after carbon dioxide inhalation. The samples were collected into tubes containing K3EDTA for evaluation of hematology parameters, sodium citrate for evaluation of coagulation parameters, and serum separators with no anticoagulant for the clinical chemistry samples. The order of bleeding was by alternating one animal from each dose group, then repeating to reduce handling and time biases. Urine samples were collected by placing the animals in stainless steel metabolism cages for at least 12 hours.

Necropsy

Macroscopic
Necropsy examinations were performed under procedures approved by a veterinary pathologist on all animals at the scheduled terminal and recovery necropsies. The animals were euthanized by carbon dioxide inhalation followed by exsanguination by transection of the abdominal vena cava. The animals were examined carefully for external abnormalities including palpable masses. The skin was reflected from a ventral midline incision and any subcutaneous masses were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities. The organs were
removed, examined, and, where required, placed in fixative. The pituitary was fixed in situ.

All designated tissues were fixed in neutral buffered formalin, except for the eye (including the optic nerve) and testes, which were fixed using a modified Davidson’s fixative1. Eyes and testes were placed into formalin following fixation. Formalin was infused into the lung via the trachea and into the urinary bladder. A full complement of tissues and organs is as follows:

- Adrenal (2)*
- Aorta
- Bone with marrow [femur]
- Bone with marrow [sternum]
- Bone marrow smear [2 collected]a
- Brain [cerebrum, midbrain, cerebellum, medulla/pons, olfactory bulbs]*
- Epididymis (2)*
- Eye including optic nerve (2)
- GALT [Gut Associated Lymphoid Tissue]
- Gastrointestinal tract:
esophagus
stomach [glandular and nonglandular]
duodenum
jejunum
ileum
cecum
colon
rectum
- Gonads:
ovary (2)*
testis (2)*
- Gross lesions
- Heart*
- Joint, tibiofemoral
- Kidney (2)*
- Liver [3 sections collected; 2 examined]*
- Lung with bronchi [collected whole; 2 sections examined]
- Lymph nodes: mandibular [2 collected; 1 examined] and mesenteric
- Mammary gland [process females only]
- Pancreas
- Pituitary*
- Prostate and seminal vesicle (2)
- Salivary gland, mandibular/sublingual [2 collected; 1 examined]
- Salivary gland, parotid [2 collected; 1 examined]
- Sciatic nerve
- Skeletal muscle, biceps femoris
- Skin
- Spinal cord [cervical, thoracic, and lumbar]
- Spleen*
- Thymus*
- Thyroid/parathyroid (2)*
- Tongue
- Trachea
- Urinary bladder
- Uterus [both horns]/Cervix*
- Vagina

a Bone marrow smears were collected at scheduled necropsies and held.
(2) Paired organ
*Weighed organ

Organ Weights
Body weights and protocol-designated organ weights were recorded for all animals at the scheduled necropsies and appropriate organ weight ratios were calculated (relative to body and brain weights). Paired organs were weighed together. A combined weight for the thyroid and parathyroid glands was collected. The thyroid/parathyroid gland and pituitary gland were weighed following fixation.

Microscopic
Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The adrenal glands, liver, lung, ovaries, thyroid gland, and uterus with cervix were determined to be potential target organs and were examined for all groups. The slides were examined by a board-certified veterinary pathologist. A four-step grading system was utilized to define gradable lesions for comparison between dose groups.
Sacrifice and pathology:
Clinical Pathology: Clinical pathology evaluations were conducted on designated animals prior to the terminal and recovery necropsies. The animals had access to drinking water but were fasted overnight prior to scheduled sample collection. Blood samples (approximately 4 mL) were collected from the vena cava after carbon dioxide inhalation. The samples were collected into tubes containing K3EDTA for evaluation of hematology parameters, sodium citrate for evaluation of coagulation parameters, and serum separators with no anticoagulant for the clinical chemistry samples. The order of bleeding was by alternating one animal from each dose group, then repeating to reduce handling and time biases. Urine samples were collected by placing the animals in stainless steel metabolism cages for at least 12 hours.

Postmortem Study Evaluations: Postmortem study evaluations were performed on all animals at the scheduled terminal and recovery necropsies.
Other examinations:
None further examinations specified in the study report.
Statistics:
Statistical Comparison and Statistcal Analysis reported in table form - See "any other information".

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight among males administered at 325 mg/kg/day was generally lower throughout the dosing and recovery intervals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was generally increased and was sporadically statistically significant during the dosing period in females administered Reofos 35 at 325 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increases in cholesterol, urea nitrogen and globulin
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
During Week 13, statistically significant decreases in fine movement (count)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
organ weight changes were present in the adrenal glands, in the liver, in the thyroid/parathyroid glands and in the ovaries in females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related macroscopic findings were present in the adrenal glands at all dose levels.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Homogeneity: Analysis indicated that the formulation technique utilized produced homogenous preparations (average percent recovery ±10% and RSD ≤5%). Tabulated data is listed below under "any other information"

Concentration: The concentration analysis results indicate the formulations were accurately prepared (results were within ± 10% of nominal).
Percent Relative Standard Deviation (%RSD) for the 100 mg/kg/day formulation prepared for Week 1, and the 325 mg/kg/day formulations prepared for Weeks 2 and 3 were outside the acceptance criterion of ≤5%; however, all the corresponding average recoveries were within 10% of the nominal concentration, therefore backup samples were not analyzed. Tabulated data is listed below under "any other information"

Mortality: All animals survived to the scheduled necropsies.

Cage side Clinical Observations: Salivation was consistently noted during the dosing period for several males and females at ≥100 mg/kg/day. Salivation was higher in incidence for animals receiving Reofos 35 at 325 mg/kg/day than for animals at 100 mg/kg/day. No other test article-related clinical signs were noted during the study.
Occasionally, scabbed areas, material around the mouth, sparse hair, hair discoloration, and/or abrasions were noted; however, these findings were not considered test article related since they either commonly occur in animals of this strain and age, were present at low incidence (i.e. 1 to 2 animals/sex/group) with no dose response pattern, or were isolated/transient incidences which were considered to be of no toxicological relevance.

Neurobehavioral Findings: There were no test article-related neurobehavioral effects noted during the study. During Week 2, convulsions were noted in a single animal administered Reofos 35 at 25 mg/kg/day; however, no corresponding findings to support this observation were noted. All other neurobehavioral endpoints for this animal evaluated at that time were within normal limits for this species. Furthermore, similar findings were not observed for any animal at any other dose level; therefore, this was considered an isolated and incidental event and not considered related to the administration of Reofos 35.

Functional Observational Battery Evaluations: There were no adverse test-article related effects on FOB parameters in males or females at any dose level evaluated during the terminal or recovery phases. Mean body weight among males administered Reofos 35 at 325 mg/kg/day was statistically significantly lower for Week 13, when compared to controls. Lower mean body weight was observed throughout the dosing period for males at 325 mg/kg/day as discussed in the body weight section below, and was considered related to the administration of Reofos 35. Salivation also was noted during the Week 13 evaluations for 2 to 3 males and/or females at ≥100 mg/kg/day.
Salivation was consistently observed during the dosing period for animals at ≥100 mg/kg/day as discussed above in the cage side clinical observations section and was considered test article-related. Statistically significant increases in mean thermal response were observed for males at 100 mg/kg/day for Week 13; however the higher mean was attributed to a single outlier (animal number 3001) and was not considered related to the administration of Reofos 35.

Locomotor Activity: During Week 13, statistically significant decreases in fine movement (count) and in total distance (cm) were noted in males at 325 mg/kg/day 10-20 minutes after placement into the chamber; however, basic and fine movement for the total monitoring time (0-60 minutes) were comparable to controls. In addition, statistically significant decreases in rearing (count) were noted in males at 325 mg/kg/day for the total duration of monitoring (0-60 minutes).
Although changes observed for rearing in males at 325 mg/kg/day may have been related administration of Reofos 35, a definitive alteration is unclear. During the recovery period, statistically significant decreases in basic and fine movement (count) were noted for females at 325 mg/kg/day 50-60 minutes after placement into the chamber; however, basic and fine movement for the total monitoring time (0-60 minutes) were comparable to controls. No other significant changes in locomotor activity were noted. All other mean values were within the normal range of variation and are therefore considered normal behavior of animals of this age and strain (MPI Research Historical Control Locomotor Activity Data Version 09-01).

Body Weights: Mean body weight among males administered Reofos 35 at 325 mg/kg/day was generally lower (4 to 13%) throughout the dosing and recovery intervals, when compared to controls.
Mean body weight for the males was statistically significantly lower from Weeks 5 to 13 (9 to 13% lower), when compared to controls, and was considered related to the administration of Reofos 35. No corresponding effects on food consumption were noted. No test article-related effects on mean body weight were noted for females at any dose level.

Food Consumption: No test article-related effects for food consumption were noted for males at any dose level.
Mean food consumption was generally increased and was sporadically statistically significant during the dosing period in females administered Reofos 35 at 325 mg/kg/day.

Ophthalmoscopic Examinations: There were no test article-related effects observed at the terminal or recovery ophthalmoscopic examinations.

Hematology: There were no test article-related effects among hematology parameters in any treatment group at the terminal collection, or in either sex administered 325 mg/kg/day at the recovery collection. Despite occasional statistical differences, all mean and individual values were considered within an acceptable range for biologic and/or procedure-related variation.

Coagulation: At the terminal collection, a mild statistically significant increase in fibrinogen (+27%) was present in males administered 325 mg/kg/day, relative to controls. The increase in fibrinogen correlated with an increase in globulin, and was most typical of an inflammatory response, but lacked correlative hematologic or microscopic findings. The increase in fibrinogen was likely test article-related, but was fully resolved at the recovery collection, and was not noted in females.
Statistically significant alterations in prothrombin time were present at the terminal collection in males (+9%) and females (-6%) administered 325 mg/kg/day, relative to controls. These changes were not considered test article-related or toxicologically meaningful due to the differing direction of the changes and small magnitude.

Clinical Chemistry: At the terminal collection, mild statistically significant increases in cholesterol were present in males administered 100 mg/kg/day (+39%) and both sexes administered 325 mg/kg/day (up to +34%), relative to controls. Increases in cholesterol were considered test article-related. These effects resolved at the recovery collection in both sexes administered 325 mg/kg/day.
At the terminal collection, mild statistically significant increases in urea nitrogen (up to +23%) were present in males administered 100 and 325 mg/kg/day, relative to controls.
Increases in urea nitrogen were considered test article-related, and were resolved at the recovery collection in males administered 325 mg/kg/day. Similar changes were not noted in females.
At the terminal collection, males administered 325 mg/kg/day had a mild statistically significant increase in globulin (+9%), relative to controls. The increase in globulin resulted in a mild statistically significant decrease in the albumin/globulin ratio (-9%), and correlated with the increase in fibrinogen, discussed previously. The increase in globulin was likely test article-related, but was not noted in females, and was fully resolved at the recovery collection.
Despite other statistical differences, all other mean and individual values were considered within an acceptable range for biologic and/or procedure-related variation.

Urinalysis: No test article-related alterations were observed among urinalysis parameters in any treatment group at the terminal collection, or in either sex administered 325 mg/kg/day at the recovery collection. There were occasional differences found in urine volume and specific gravity that were not considered toxicologically meaningful due to their sporadic nature and the inherent variability of these endpoints. There were minor variations between treatment groups among pH, and biochemical (protein, glucose, bilirubin, etc.) urinary components; however, all findings were considered within an acceptable range for biologic and/or procedure-related variability.

Unscheduled Deaths: All animals survived to the scheduled necropsies.

Macroscopic: At the terminal necropsy, test article-related macroscopic findings were present in the adrenal glands at all dose levels. Tan discoloration and enlargement of the adrenal glands were considered test article related at 25, 100, and 325 mg/kg/day (see table). The changes correlated to diffuse vacuolation of the zona fasciculata. Following the recovery period, none of these changes were noted.
All other macroscopic findings noted in the study are commonly encountered in Sprague Dawley rats and were considered incidental. Tabulated data is listed below under "any other information"

Organ Weights: At the terminal necropsy, test article-related organ weight changes were present in the adrenal glands in males at 100 and 325 mg/kg/day and in females at all dose levels, in the liver in males and females at 100 and 325 mg/kg/day, in the thyroid/parathyroid glands in males at 325 mg/kg/day, and in the ovaries in females at all dose levels. Following the recovery period, adrenal gland weights in males and ovary weights in females were slightly higher compared to controls. The liver and thyroid gland weights were similar to controls.
At the terminal necropsy, dosing with the test article resulted in lower final body weights in males at 325 mg/kg/day which resulted in higher organ weights relative to body weights, lower absolute organ weights, and/or lower organ weights relative to brain weights in the following organs: brain, epididymides, heart, and pituitary gland.
Other organ weight differences were considered the result of normal biological variation based on the lack of dose response, the minimal magnitude, and/or the absence of microscopic correlates. Tabulated data is listed below under "any other information"

Microscopic: Test article-related microscopic changes were present in the adrenal glands in males and females at all dose levels (diffuse vacuolation of the zona fasciculata and glomerulosa), in the ovaries in females at all dose levels (interstitial cell vacuolation), in the liver (panlobular or centrilobular hypertrophy) in males at all dose levels and in females at 325 mg/kg/day, and in the thyroid gland (follicular cell hypertrophy) in males at 325 mg/kg/day and in females at 100 and 325 mg/kg/day. Adrenal gland changes were considered adverse at all dose levels based on their severity and the marked increases in weights noted in females. All changes were completely reversible or showed evidence of reversibility following the recovery period.
Diffuse vacuolation of the adrenal glands were present in all animals dosed with the test article. The change was minimal to severe and the severity was dose dependent (see table). The change was limited to the zona fasciculata and zona glomerulosa and was characterized by enlarged cells with foamy cytoplasm. In addition, the zona fasciculata cells present near the zona reticularis had large single clear vacuoles that expanded outwardly based on severity. This finding resulted in enlarged adrenal glands and/or tan discoloration of the adrenal glands at necropsy and higher adrenal gland weights. Following the recovery period, the diffuse vacuolation was not present but cells near the zona reticularis had large single cytoplasmic vacuoles (identified as increased vacuolation). Based on the magnitude of the organ weight increases in females, the finding was considered adverse at all dose levels.
Most females dosed with the test article had interstitial cell vacuolation in the ovaries and fewer females had increased corpus luteum in the ovaries at 100 and 325 mg/kg/day.
Interstitial cell vacuolation was graded minimal to moderate and the severity was dose related (see incidence table). The change was characterized by enlarged pale round stromal cells with clear vacuolated cytoplasm. In addition, PCNA (proliferating cell nuclear antigen) was detected in some of these cells indicative of a proliferative change. The vacuolation was associated with slight increases in ovary weights at all dose levels.
Following the recovery period, the change was still present in 4/5 females at 325 mg/kg/day, albeit with a lower severity, indicating slow reversibility.
Panlobular hepatocellular hypertrophy of the liver was considered test article related in males at all dose levels (see incidence table). The change was characterized by a diffuse minimal enlargement of hepatocytes affecting the whole lobule. In contrast, hypertrophy was limited to the centrilobular area in females at 325 mg/kg/day only. The hepatocellular hypertrophy correlated to higher liver weights in males and females at 100 and 325 mg/kg/day. The change was completely reversible following the recovery period and the finding was not considered adverse based on the minimal severity and the slight increase in liver weights.
Follicular cell hypertrophy of the thyroid glands was present in 1/10 females at 100 mg/kg/day, and 2/10 males and 4/10 females at 325 mg/kg/day at the terminal necropsy and 1/5 males at 325 mg/kg/day at the recovery necropsy. The change was characterized by tall cuboidal follicular cells with vacuolated cytoplasm. Follicles were also reduced in size with little to no colloid. The change was associated with minimally higher weights in males at 325 mg/kg/day only (terminal necropsy) and was not considered adverse.
All other microscopic changes noted are commonly encountered in Sprague Dawley rats and were considered incidental. Tabulated data is listed below under "any other information"

Effect levels

Dose descriptor:
NOAEL
Effect level:
< 25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on adverse histopathological changes observed in the adrenal glands for males and females at all dose levels.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Homogeneity

Analysis indicated that the formulation technique utilized produced homogenous preparations (average percent recovery ±10% and RSD ≤5%).

Homogeneity Analysis of Reofos 35- Week 1

Dose Level (mg/kg)

Nominal Concentration (mg/mL)

Average Calculated Concentration (mg/mL)

Average %Recoverya

%Relative Standard Deviation

25

5.9

4.73

94.5

1.084

325

65.0

66.05

101.6

5.195

aAverage %recovery was calculated from the nominal concentration

 

Concentration

The concentration analysis results indicate the formulations were accurately prepared (results were within ± 10% of nominal). Percent Relative Standard Deviation (%RSD) for the 100 mg/kg/day formulation prepared for Week 1, and the 325 mg/kg/day formulations prepared for Weeks 2 and 3 were outside the acceptance criterion of ≤5%; however, all the corresponding average recoveries were within 10% of the nominal concentration, therefore backup samples were not analyzed.

Concentration Analysis of Reofos 35- Weeks 1, 2, 3, 4, 8 and 12

Dose Level (mg/kg)

Nominal Concentration (mg/mL)

Average Calculated Concentrationa(mg/mL)

Average %Recoverya,b

%Relative Standard Deviationa

0

0

BLQ

NA

NA

25

5.0

4.73 – 5.07

94.6 – 104.5

0.040 – 2.764

100

20.0

18.50 – 20.64

92.5 – 103.2

0.027 – 6.664

325

65.0

61.89 – 66.05

95.2 – 103.2

0.347 – 12.746

aResults are the range of values determined during Weeks 1, 2, 3, 4, 8 and 12.

bAverage %recovery was calculated from the nominal concentration

NA – Not Applicable

BLQ – Below the limit of quantification

 

Macroscopic

Test Article-related Macroscopic Observation – Terminal

Dose Level: mg/kg/day

0

25

100

325

Sex

M

F

M

F

M

F

M

F

Number Examined

10

10

10

10

10

10

10

10

Adrenal glands

Discoloration, tan

 

-mild

0

0

0

1

1

1

0

4

Enlarged

0

0

0

1

0

3

0

2

 

-minimal

0

0

0

0

0

0

0

1

 

-mild

0

0

0

1

0

3

0

1

M – Male

F - Female

 

Organ Weights

Test Article-related Organ Weight Changes – Terminal

Male and Female (Percent change relative to control)

Dose level: mg/kg/day

25

100

325

Sex

M

F

M

F

M

F

Number Examined

10

10

10

10

10

10

Adrenal glands (g)

↑6.33

↑47.83b

↑25.32

↑75.36b

↑46.84b

↑98.55b

Adrenal glands/BWt%

↑8.66

↑49.78b

↑22.05

↑81.78b

↑80.31b

↑103.11b

Adrenal glands/BWt ratio

↑8.40

↑51.29b

↑22.22

↑73.07b

↑52.57b

↑104.01b

Liver (g)

↑7.84

↑4.01

↑27.13a

↑11.33a

↑19.08

↑30.69b

Liver/BWt%

↑9.04

↑7.06

↑22.35b

↑15.30b

↑43.75b

↑33.81b

Liver/BrWt ratio

↑9.12

↑6.76

↑23.80a

↑9.16

↑23.36a

↑33.55b

Ovaries (g)

NA

↑20.00

NA

↑16.84

NA

↑33.68

Ovaries/BWt%

NA

↑23.45

NA

↑24.10

NA

↑37.46a

Ovaries/BrWt ratio

NA

↑23.96

NA

↑14.58

NA

↑36.88a

Thyroid/parathyroid glands (g)

0.00

↓12.50

↑6.90

↑8.33

↑3.34

↑4.17

Thyroid/parathyroid glands/BWt%

0.00

↓11.54

↑2.08

↑6.41

↑22.92a

↑8.97

Thyroid/parathyroid glands/BrWt ratio

↓0.72

↓12.30

↑2.17

↓11.48

↑4.35

↑8.20

aSignificantly different from control; (p<0.05)

bSignificantly different from control; (p<0.01)

BWt – Bodyweight

BrWt – Brain Weight

NA – Not Applicable

↑ - Increased

↓ - Decreased

M – Male

F - Female

 

Microscopic

Test Article-related Microscopic Observations – Terminal

Dose level: mg/kg/day

0

25

100

325

Sex

M

F

M

F

M

F

M

F

Number Examined

10

10

10

10

10

10

10

10

Adrenal glands

Vacuolation, diffuse

0

0

10

10

10

10

10

10

 

-minimal

0

0

3

7

0

0

0

0

 

-mild

0

0

3

3

2

5

1

1

 

-moderate

0

0

4

0

7

5

7

9

 

-severe

0

0

0

0

1

0

2

0

Vacuolation, increased

 

 

 

 

 

 

 

 

 

-minimal

4

0

0

0

0

0

0

0

M- Male

F - Female

 

Test Article-related Microscopic Observations – Recovery

Dose level: mg/kg/day

0

325

Sex

M

F

M

F

Number Examined

5

5

5

5

Adrenal glands

Vacuolation, increased

0

0

5

5

 

-minimal

0

0

0

3

 

-mild

0

0

1

2

 

-moderate

0

0

3

0

 

-severe

0

0

1

0

M – Male

F – Female

 

Test Article-related Microscopic Observations – Terminal

Female

Dose level: mg/kg/day

0

25

100

325

Number Examined

10

10

10

10

Ovaries

Corpus luteum increased

 

 

 

 

 

-minimal

0

0

1

4

Vacuolation, interstitial cell

0

9

9

10

 

-minimal

0

5

0

0

 

-mild

0

4

4

3

 

-moderate

0

0

5

7

 

Test Article-related Microscopic Observations – Recovery

Female

Dose level: mg/kg/day

0

325

Number Examined

5

5

Ovaries

 

 

Vacuolation, interstitial cell

0

4

 

-minimal

0

2

 

-mild

0

2

 

Test Article-related Microscopic Observations – Terminal

Dose level: mg/kg/day

0

25

100

325

Sex

M

F

M

F

M

F

M

F

Number Examined

10

10

10

10

10

10

10

10

Liver

 

 

 

 

 

 

 

 

Hypertrophy, hepatocellular, panlobular

 

 

 

 

 

 

 

 

 

-minimal

0

0

1

0

2

0

9

0

Hypertrophy, hepatocellular, centrilobular

 

 

 

 

 

 

 

 

 

-minimal

0

0

0

0

0

0

0

2

M – Male

F - Female

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, where Reofos 35 was administered via oral gavage for 91 consecutive days to male and female rats at 0, 25, 100, and 325 mg/kg/day, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be less than 25 mg/kg/day (lowest dose administered) based on adverse histopathological changes observed in the adrenal glands for males and females at all dose levels. Macroscopic and organ weight changes in the adrenal gland correlated to diffuse vacuolation of the zona fasciculata and were characterized by enlarged cells with foamy cytoplasm. In addition, the zona fasciculata cells present near the zona reticularis had large single clear vacuoles that expanded outwardly depending on severity. Adversity was based on the severity, and the increases in weights noted in females. At recovery the diffuse vacuolation was not present; however, cells near the zona reticularis had large single cytoplasmic vacuoles (identified as increased vacuolation).
Executive summary:

This study was conducted to evaluate the potential toxicity of the test article, Reofos 35, when administered to rats for 91 days via oral gavage and to evaluate the reversibility of any effects following a 28-day test article-free recovery period.

The study was conducted in accordance with MPI Research Standard Operating Procedures (SOPs) and the protocol as approved by the Sponsor. In the opinion of the Study Director, none of the deviations affected the quality or integrity of the study. This study was based on the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011; Test Guideline 408, OECD Guideline for the Testing of Chemicals, revised September 1998; and United States EPA, Office of Prevention, Pesticides, and Toxic Substances (OPPTS), Guideline 870.3100, 90-Day oral toxicity in rodent, August 1998.

 

Assessments of neurobehavioral effects and systemic toxicity were based on mortality, clinical signs, body weight, food consumption, functional observational battery (FOB) evaluations, locomotor activity, ophthalmoscopic and neurobehavioral examinations, and clinical and anatomic pathology.

 

There were no adverse test article-related effects noted for any of the in-life parameters evaluated. Test article-related clinical pathology changes observed included: reversible increases in cholesterol for males at 100 mg/kg/day and in both sexes at 325 mg/kg/day; reversible increases in fibrinogen and globulin for males at 325 mg/kg/day (suggestive of an inflammatory response); and reversible increases in urea nitrogen for males at ≥100 mg/kg/day. No test article-related effects were observed for males or females at 25 mg/kg/day for the aforementioned parameters.

 

Macroscopic findings noted at the terminal necropsy included tan discoloration and enlargement of the adrenal glands at all Reofos 35 dose levels. Test article-related adrenal gland weight changes were present in males at ≥100 mg/kg/day and in females at all dose levels. Macroscopic and organ weight changes correlated to diffuse vacuolation of the zona fasciculata observed microscopically and characterized by enlarged cells with foamy cytoplasm. In addition, the zona fasciculata cells present near the zona reticularis had large single clear vacuoles that expanded outwardly depending on severity. The adrenal gland changes were considered adverse test article-related effects based on the severity and the marked increases in weights noted in females. At recovery, the diffuse vacuolation was not present; however, cells near the zona reticularis had large single cytoplasmic vacuoles (identified as increased vacuolation).

 

Test article-related organ weight changes were also present in the liver for both sexes at ≥100 mg/kg/day, in the thyroid glands for males at 325 mg/kg/day, and in the ovaries for females at all dose levels. Liver, thyroid gland, and ovary organ weight changes correlated microscopically to centrilobular or pablobular hypertrophy in the liver, follicular cell hypertrophy in the thyroid glands, and interstitial cell vacuolation in the ovaries. Additionally, lower terminal body weight in males at 325 mg/kg/day resulted in higher organ weight relative to body weights, lower absolute weights, and/or lower organ weights relative to brain weights for the brain, epididymides, heart, and the pituitary gland. These changes were also considered related to the administration of Reofos 35.

 

Under the conditions of this study, where Reofos 35 was administered via oral gavage for 91 consecutive days to male and female rats at 0, 25, 100, and 325 mg/kg/day, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be less than 25 mg/kg/day (the lowest dose administered) based on adverse histopathological changes observed in the adrenal glands for males and females at all dose levels.