Registration Dossier

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:
September 24, 2014 to December 23, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & US 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:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The following unplanned deviations occurred as the result of unintended deviations from the protocol.On the day of animal receipt, the male animals were less than 6 weeks of age at arrival.On the day of animal receipt, the males had a body weight range of 121 to 149 g, which was outside the protocol-specified body weight range of 130 g to 210 g. This range is provided in order to ensure that animals of similar weight, age, and condition are used for study. Despite this deviation, these animals were adequately similar to accomplish the objectives of the 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:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear yellow liquid
Details on test material:
Label Identification: Hatcol 3346
Lot Number: 2013059135
Physical Characteristics: Clear yellow liquid
Retest Date: May 30, 2017
Storage: Room temperature, protected from light
TMC Number: 1405A8 & 1409FC
Viscosity (40°C): 44 cSt
Viscosity (100°C): 6.2 cSt
Pour Point: 0°C
Flash Point: 249°C
Total Acid Number: 0.01 mgKOHg
Water Content: 35ppm
Density (15.6°C): 8.04 lb/USG

Test animals

Species:
rat
Strain:
other: CD® [Crl:CD®(SD)]
Sex:
male/female
Details on test animals and environmental conditions:
Animal Acquisition and AcclimationA total of 46 male and 46 female CD® [Crl:CD®(SD)] rats (approximately 6 weeks of age) were received from Charles River Laboratories, Stone Ridge, New York, on September 16, 2014. During the 8-day acclimation period, the animals were observed daily with respect to general health and any signs of disease. The week prior to dose initiation, animals were administered a sham dose of tap water on two occasions in the same manner intended for use on study.Randomization, Assignment to Study, and MaintenanceUsing a standard, by weight, measured value randomization procedure, 40 male and 40 female animals (weighing 192 to 224 g and 145 to 187 g, respectively, at randomization) were assigned to the control and treatment groups identified in table form (see Any other information).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 transferred to an MPI Research training colony.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 two per cage (same sex) in solid bottom cages with nonaromatic bedding in an environmentally controlled room. Animal enrichment was provided according to MPI Research SOP. Fluorescent lighting was provided for approximately 12 hours per day. The dark cycle was interrupted intermittently due to fasting for clinical pathology evaluations. 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. The actual temperature and humidity findings are not reported but are maintained in the study file.Block Lab Diet® (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum, except during periods of fasting for clinical pathology blood collections. The lot number from each diet lot used for this study was recorded. Certification analysis of each diet lot was performed by the manufacturer. Tap water was available ad libitum via an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to MPI Research SOP. The results of food and water analyses are retained in the Archives. 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. Therefore, no analyses other than those stated above were conducted.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Label Identification: Corn oil Lot Number: 2DD0215 Physical Characteristics: Clear yellow liquid Expiration Date April 30, 2015 Storage: Room temperature, protected from light TMC Number: CANOIL14 (also known as 140586)
Details on oral exposure:
The test article, Hatcol® 3346, 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 under yellow lighting by mixing the test article with an appropriate amount of vehicle using a magnetic stir bar and stir plate to achieve the nominal concentrations of 20, 60, and 200 mg/mL, and were stored at room temperature and protected from light in amber glass bottles.The vehicle and test article were administered daily for 90 consecutive days via oral gavage. The dose levels were 100, 300, and 1000 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 prior to administration. 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:
Analysis of Dosing Formulations: Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples (see table below under "additional information") were collected while stirring, using a positive displacement pipette, and placed into 20 mL amber glass scintillation vials.Formulations were analysed as follows:Analyses: Samples (including back up samples) were stored at room temperature and protected from light until analyzed.Objective: To determine the homogeneity and concentration of Hatcol® 3346 in dosing formulations.Compliance: This nonclinical laboratory study phase was conducted in accordance with the United States Environmental Protection Agency (EPA), Toxic Substance Control Act (TSCA) Good Laboratory Practice (GLP) Standards, 40 Code of Federal Regulations (CFR) Part 792, the Organization for Economic Cooperation and Development (OECD) Principles on GLP [as revised 1997; ENV/MC/CHEM(98)17], and the United States EPA, Federal Insecticide, Fungicide and Rodenticide ACT (FIFRA), GLP Standards, 40 CFR Part 160.Analytical Method Number and Title: 1038-049: Determination of Hatcol® 3346 in Corn Oil Dose Formulations by HPLC/UVReference Standard: Hatcol® 3346Batch Number: 2013059135MPI Research Inventory ID: 1405A8 (SP5003085)Storage: Room temperature, protected from lightCorrection Factor: noneData Collection and Analysis Software: Empower™ 2: Build 2154 (Waters Technologies Corporation®); ExyLIMS Version 3.0 (MPI Research, Mattawan, Michigan)HPLC Conditions: Liquid chromatography system equipped with an Ascentis Express RP-Amide column, 4.6 x 100 mm, 2.7 μm particle size with a gradient flow of 0.1% phosphoric acid in water (mobile phase A) and 0.1% phosphoric acid in acetonitrile (mobile phase B) at a flow rate of 1.5 mL/minute.Analysis Description: Prior to analysis, duplicate samples were diluted with isopropyl alcohol (diluent) to within the range of the calibration curve. Vehicle samples were diluted with diluent using a dilution factor of 100. An aliquot of each sample was injected into the HPLC-UV system for analysis and evaluated at 225 nm.Regression Type: Linear, unweightedStudy Sample Receipt(s): Number of Samples: 168Date(s) of receipt by Analytical Department: September 23, 2014 to December 9, 2014Study Sample Storage Conditions: Ambient, protected from lightStorage Stability: All samples were analyzed within the established storage stability determined under MPI Research Study Number 1038-049(1).Run Acceptance CriteriaSystem Suitability Test Standards: 1. Injection repeatability (peak area and retention time) ≤2% RSD (relative standard deviation)2. Resolution between the analyte peak and any adjacent peaks must be ≥1.53. Tailing Factor (T) ≤24. Theoretical Plates (N) ≥2000Calibration Standards: 1. Accuracy within ±5% of the nominal concentration2. Coefficient of determination (R2) ≥0.995 Performance Check Standards (Same preparation as the System Suitability Standard):1. Periodically injected so that no more than 10 samples are bracketed by performance check standards. Samples are considered valid if bracketed by passing performance check standard injections.2. Accuracy within ±5% of the nominal concentrationBlank Injections: ≤20% of the effective limit of quantitation (ELOQ)AssessmentsHomogeneity: 1. Average concentration within ±10% of the nominal concentration2. Precision ≤5% RSDConcentration: 1. Average concentration within ±10% of the nominal concentration2. Precision ≤5% RSD3. Vehicle (control) samples < ELOQConclusion: A total of 64 samples were analyzed for Hatcol® 3346 in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results.
Duration of treatment / exposure:
The vehicle and test article were administered for 90 consecutive days via oral gavage.
Frequency of treatment:
The vehicle and test article were administered daily.
Doses / concentrations
Remarks:
Doses / Concentrations:0, 100, 300, 1000 mg/kg/dayBasis:actual ingested
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Justification of Test SystemThe state of scientific knowledge at the time of study initiation and the applicable guidelines cited previously did not provide acceptable alternatives, in vitro or otherwise, to the use of live animals to accomplish the purpose of this study. “The development of knowledge necessary for the improvement of the health and well-being of humans as well as other animals requires in vivo experimentation with a wide variety of animal species.” “Whole animals are essential in research and testing because they best reflect the dynamic interactions between the various cells, tissues, and organs comprising the human body.” The rat is the usual rodent model used for evaluating the toxicity of various classes of chemicals and for which there is a large historical database. The rat is the required species designated in the regulatory guidelines for this 90-day study.Justification for Route of AdministrationThe oral route is one of the potential routes of human exposure to this test article.Justification of Dose LevelsThe dose levels were selected on the basis of available data from a 28-day range finding toxicity study (MPI Research Study Number 1038-046). No signs of systemic toxicity were evident at the highest dose level evaluated. The high dose, 1000 mg/kg/day, represents the limit dose required by the applicable testing guidelines. The low and mid-dose levels were chosen to provide a grade response.AdministrationThe vehicle and test article were administered daily for 90 consecutive days via oral gavage. The dose levels were 100, 300, and 1000 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 prior to administration. Individual doses were based on the most recent body weights.
Positive control:
Positive control not required for this study.

Examinations

Observations and examinations performed and frequency:
In-life ExaminationsCage side ObservationsAll animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily. On occasion, veterinary consultations were conducted during the course of the study for health monitoring purposes (i.e. hind limb impairment or thin body condition). All treatments and observations were recorded. The medical treatments and observations are not reported but are maintained in the study file.Daily Cage side Clinical ObservationsCage side clinical observations were conducted on each animal once daily except on days when detailed clinical observations and neurobehavioral observations were required.Each animal was examined visually while still in the cage for clinical signs of disease, toxicity, and injury. If warranted, the animal was removed from the cage for further signs of disease, toxicity, and injury. The persistence or disappearance of findings was documented at the next scheduled observation. The animals were observed outside the cage if scheduled observations occur concurrently with other study related activities.Detailed Clinical ObservationsA detailed clinical examination of each animal was performed prior to initiation of test article administration and once weekly thereafter. 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, lacrimation, piloerection, and pupil size.Neurobehavioral EvaluationsNeurobehavioral evaluations were conducted on each animal prior to test article administration and once weekly thereafter.Observations were made outside the animal’s home cage in a standard arena (with the exception of posture and activity level) using an applicable scoring system at approximately the same time of day (i.e., morning or afternoon), each week. Observations for posture and activity level were made inside the animal’s home cage. 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 gait, posture, and reactivity to handling, activity level/movement, as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling), or bizarre behaviour (e.g., self-mutilation, walking backwards) were also recorded.Functional Observational Battery ObservationsFunctional 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 at 1 hour postdose. 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 hind limb grip strength was measured using the procedure described by Meyer, et al., and hind limb 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 as described by Ankier. Body weight and temperature were also measured. Locomotor ActivityLocomotor activity evaluations were conducted on all animals from 1 to 3 hours postdose during Week 13 of test article administration. 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 WeightsBody weights for all animals were measured and recorded at receipt, prior to randomization on Day -1 and once weekly thereafter. Food ConsumptionFood consumption was measured and recorded weekly during the study.Ophthalmoscopic ExaminationsOphthalmoscopic examinations were conducted on all animals pretest and prior to termination by Joshua T. Bartoe, D.V.M., D.A.C.V.O.Clinical PathologyClinical pathology evaluations were conducted on all animals prior to terminal necropsy.The animals had access to drinking water but were fasted (food removed) overnight prior to scheduled sample collection. Blood samples (approximately 4 mL) were collected via 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.
Sacrifice and pathology:
Post-mortem Study EvaluationsPost-mortem study evaluations were performed on all animals euthanized at the scheduled necropsy as follows:1.1. MacroscopicNecropsy examinations were performed under procedures approved by a veterinary pathologist on all animals at the scheduled necropsy. 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.1.2. Organ WeightsBody weights and protocol-designated organ weights were recorded for all animals at the scheduled necropsy and appropriate organ weight ratios were calculated (relative to body and brain weights). Paired organs were weighed together.1.3. MicroscopicMicroscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The lung and thyroid gland were determined to be potential target organs and were examined for the 100 and 300 mg/kg/day 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.
Other examinations:
No further examinations detailed in the study report.
Statistics:
The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group using the analysis outlined below. Data for some endpoints, as indicated, were transformed by either a log or rank transformation prior to conducting the specified analysis. The experimental unit for the analysis of food consumption was the cage. Statistical Comparisons and Statistical Analysis detailed 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:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
HomogeneityResults of the dosing formulation analysis, expressed as % recovery of the nominal concentration, ranged from 99.7 to 102.5%. All Hatcol® 3346 dosing solutions were homogeneous under conditions of use in this study (Average Percent Recovery 100±10% and RSD ≤5%).ConcentrationDose formulation concentration results, expressed as % recovery of the nominalconcentration, ranged from 97.0 to 109.5%. All Hatcol® 3346 dosing solutions wereaccurately formulated (i.e., within 10% of the nominal concentration). In addition, the testarticle was not detected in the vehicle control formulation.In-life ExaminationsMortalityAll animals survived to the scheduled necropsy.Cageside and Detailed Clinical ObservationsThere were no test article-related clinical findings noted for males or females at any dose level. Occasionally, scabbed areas, sparse hair, material around the nose, hair discoloration, vocalization, limb function impairment, and/or thin body condition 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 EvaluationsThere were no test article-related neurobehavioral effects noted for males or females at any dose level. Occasionally, sporadic variations were noted over the course of the study for grid activity count and/or handling reactivity for males and/or females at all treated levels; however, due to the lack of other corresponding findings, and concurrent variations in control animals, these variations were not considered a test article-related effect.Functional Observational BatteryThere were no test-article related effects on FOB parameters in males or females at any dose level when compared to controls. Sporadic variations and one statistically significant difference in sensorimotor observations (i.e. thermal response, tail pinch response), and/or neuromuscular parameters (i.e. mean hind limb grip strength) were noted during Week 13 when compared to controls; however, due to the lack of dose dependency and/or lack of other corresponding findings, these observations were considered incidental and not test article related.Locomotor ActivityThere were no test article-related effects on locomotor activity for males or females at any dose level, when compared to controls. All mean values were within the normal range of variation and were therefore considered normal behavior of animals of this age and strain.Body WeightsThere were no test article-related body weight changes for males or females at any dose level.Food ConsumptionThere were no test article-related effects on food consumption for males or females at any dose level.Ophthalmoscopic ExaminationsThere were no test article-related effects observed at the terminal ophthalmoscopic examination.Clinical PathologyOnce daily oral administration of Hatcol® 3346 at 100, 300 or 1000 mg/kg/day for up to 90 consecutive days to rats was associated with the following non-adverse effects on clinical pathology endpoints:- Prolongations in APTT in males administered ≥100 mg/kg/day.- Increases in neutrophils in males administered 1000 mg/kg/day that lacked correlative findings in other study endpoints.- Decreases in red cell mass and increases in platelets in females administered 1000 mg/kg/day.- Decreases in potassium and phosphorus and increases in total protein, albumin and globulin in females administered 1000 mg/kg/day that lacked correlative findings in other study endpoints.HematologyAt the terminal collection, females administered 1000 mg/kg/day had statistically significant minimal decreases in red cell mass (erythrocytes, hemoglobin and hematocrit) (up to -7%), relative to controls. Decreases in red cell mass were potentially test article-related, but were not associated with meaningful alterations in absolute reticulocytes or erythrocyte morphology.A statistically significant mild increase in platelets was also present at the terminal collection in females administered 1000 mg/kg/day (+13%), relative to controls. The increase in platelets was potentially test article-related, but was not noted in males.At the terminal collection, males administered 1000 mg/kg/day had a statistically significant mild increase in neutrophils (+42%), relative to controls. Increases in neutrophils were potentially test article-related, but lacked correlative findings in other study endpoints, and were not present in females.All other fluctuations among individual and mean values, including mean values that were statistically different from controls, were considered sporadic, consistent with biologic variation, and/or negligible in magnitude, and therefore not related to test article administration.CoagulationAt the terminal collection, statistically significant and dose-dependent mild prolongations in APTT were present in males administered ≥100 mg/kg/day (up to +21%; prolonged by up to 3.23 seconds), relative to controls. With the exception of a statistically significant mild prolongation in APTT in females administered 100 mg/kg/day (+15%; prolonged 2.22 seconds), prolongations in APTT were not observed in females. In males, prolongations in APTT were considered test article-related. Due to the relatively sporadic nature of the prolongation in APTT in females at 100 mg/kg/day, it was not clearly test article-related.Clinical ChemistryAt the terminal collection, females administered 1000 mg/kg/day had statistically significant mild decreases in potassium (-14%) and phosphorus (-12%), and statistically significant mild increases in total protein (+11%), albumin (+9%) and globulin (+13%), relative to controls.These changes were potentially test article-related, but lacked correlative findings in other study endpoints (alterations in food consumption, etc.), and were not noted in males.All other fluctuations among individual and mean values, including mean values that were statistically different from controls, were considered sporadic, consistent with biologic variation, and/or negligible in magnitude, and not related to test article administration.Postmortem Study EvaluationsMacroscopicThere were no test article-related macroscopic changes.All macroscopic findings noted in the study are commonly encountered in Sprague Dawley rats and were considered incidental.Organ WeightsThere were no test article-related organ weight changes.Adrenal gland weights (absolute and relative to brain weights) were statistically higher in females at 100 mg/kg/day compared to controls. This change was considered the result of normal biological variation based on the lack of dose relationship and the minimal magnitude.MicroscopicTest article-related microscopic changes were present in the lungs in males and females at all dose levels and in the thyroid glands in males at 1000 mg/kg/day and in females at 300 and 1000 mg/kg/day. Changes consisted of vacuolated macrophages in the lungs and follicular cell hypertrophy in the thyroid glands. These findings were not considered adverse based on the minimal to mild severity.The increased incidence of minimal to mild vacuolated macrophages in the lungs was considered test article related in males and females at all dose levels.The incidence and severity were dose related. The change was graded mild when aggregates of vacuolated macrophages in the alveoli were associated with increased lymphocytic infiltrates and/or thickening of the alveolar septa. The finding was not considered adverse based on the limited severity and the absence of correlating clinical observations.Minimal follicular cell hypertrophy of the thyroid gland was considered test article related in 3/10 males at 1000 mg/kg/day and 2/10 females at 300 and 1000 mg/kg/day. The finding was characterized by smaller follicles lined by tall columnar epithelium that was occasionally vacuolated. The finding was not considered adverse based on the minimal severity.An amphophilic vacuolar renal tubule adenoma was present in 1/10 males at 300 mg/kg/day. This type of tumor is commonly encountered in Sprague-Dawley rats and the change was considered incidental based on its unique occurrence and the lack of dose response.The other microscopic changes noted in the study are commonly encountered in Sprague Dawley rats and were considered incidental.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse signs of systemic toxicity or any signs of neurotoxicity were evident.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

   

Test Article-related Microscopic Observations

Dose level: mg/kg/day

0

100

300

1000

Sex

M

F

M

F

M

F

M

F

Number Examined

10

10

10

10

10

10

10

10

Lung

               Macrophages, vacuolated

                               -minimal

                               -mild

 

0

0

0

 

3

3

0

 

5

4

1

 

7

5

2

 

6

3

3

 

10

8

2

 

10

4

6

 

10

5

5

M – Males; F - Female

  

Summary of Hematology Values - MALE

Endpoint

Study Interval

0 mg/kg/day

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Leukocytes

103/μL

Terminal

12.75

3.056

10

13.20

2.997

10

12.77

1.775

10

13.09

1.845

10

Erthrocytes

106/μL

Terminal

9.222

0.3169

10

8.880

0.3122

10

9.028

0.3064

10

8.973

0.4333

10

Hemoglobin

g/dL

Terminal

16.55

0.389

10

15.75a

0.700

10

16.36

0.782

10

15.65b

0.620

10

Hematocrit

%

Terminal

53.13

1.394

10

50.34a

2.139

10

52.16

2.482

10

50.78a

2.202

10

MCV

fL

Terminal

57.64

1.046

10

56.69

1.185

10

57.78

1.483

10

56.62

1.474

10

MCH

pg

Terminal

17.95

0.538

10

17.75

0.357

10

18.11

0.734

10

17.45

0.477

10

MCHC

g/dL

Terminal

31.16

0.638

10

31.31

0.661

10

31.36

0.779

10

30.82

0.374

10

Platelets

103/μL

Terminal

1043.0

62.90

10

1026.7

62.81

10

1014.4

99.68

10

1078.2

87.12

10

Absolute Reticulocytes

103/μL

Terminal

169.52

23.934

10

165.18

32.418

10

176.76

33.531

10

170.07

26.169

10

Neutrophils

103/μL

Terminal

1.134

0.2193

10

1.400

0.4239

10

1.310

0.2338

10

1.607a

0.4599

10

Lymphocytes

103/μL

Terminal

11.072

2.9309

10

11.240

2.7438

10

10.893

1.5977

10

10.925

1.5894

10

Monocytes

103/μL

Terminal

0.213

0.0741

10

0.233

0.0821

10

0.226

0.0715

10

0.249

0.0752

10

Eosinophils

103/μL

Terminal

0.110

0.0380

10

0.114

0.0353

10

0.117

0.0298

10

0.099

0.0307

10

Basophils

103/μL

Terminal

0.079

0.0185

10

0.077

0.0250

10

0.066

0.0107

10

0.079

0.0218

10

Other Cells

103/μL

Terminal

0.146

0.0369

10

0.112

0.0312

10

0.147

0.0472

10

0.142

0.0487

10

Summary of Hematology Values - FEMALE

Endpoint

Study Interval

0 mg/kg/day

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Leukocytes

103/μL

Terminal

9.05

1.829

10

9.43

1.673

10

9.65

1.977

10

10.64

2.881

10

Erthrocytes

106/μL

Terminal

8.459

0.3737

10

8.251

0.4154

10

8.384

0.2731

10

7.912b

0.3000

10

Hemoglobin

g/dL

Terminal

15.63

0.696

10

15.42

0.790

10

15.66

0.743

10

14.51b

0.418

10

Hematocrit

%

Terminal

49.25

1.770

10

48.32

2.599

10

49.38

1.539

10

45.85b

1.449

10

MCV

fL

Terminal

58.22

1.532

10

58.59

1.673

10

58.89

1.313

10

57.95

1.371

10

MCH

pg

Terminal

18.50

0.627

10

18.71

0.615

10

18.66

0.760

10

18.35

0.513

10

MCHC

g/dL

Terminal

31.75

0.515

10

31.95

1.080

10

31.69

1.015

10

31.67

0.517

10

Platelets

103/μL

Terminal

1073.9

105.80

10

1115.6

78.83

10

1059.2

116.02

10

1211.7a

137.89

10

Absolute Reticulocytes

103/μL

Terminal

150.66

19.395

10

156.15

32.485

10

180.48

43.894

10

153.55

34.454

10

Neutrophils

103/μL

Terminal

0.619

0.1318

10

0.771

0.5514

10

0.780

0.1864

10

0.782

0.2645

10

Lymphocytes

103/μL

Terminal

8.033

1.6851

10

8.226

1.9080

10

8.463

1.8587

10

9.378

2.7167

10

Monocytes

103/μL

Terminal

0.160

0.0548

10

0.186

0.079

10

0.185

0.0552

10

0.191

0.0470

10

Eosinophils

103/μL

Terminal

0.079

0.0409

10

0.079

0.0303

10

0.068

0.0322

10

0.067

0.0216

10

Basophils

103/μL

Terminal

0.050

0.0156

10

0.054

0.0184

10

0.063

0.0149

10

0.060

0.0316

10

Other Cells

103/μL

Terminal

0.098

0.0533

10

0.105

0.0363

10

0.094

0.0280

10

0.155

0.1183

10

MCV – Mean Corpuscular Volume                       N – Number of measures used to calculate mean           aSignificantly different from control; (p<0.05)

MCH – Mean Corpuscular Hemoglobin                 SD – Standard Deviation                                     bSignificantly different from control; (p<0.01)

MCHC – Mean Corpuscular Hemoglobin Concentration

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, where Hatcol® 3346 was administered via oral gavage for 90 consecutive days to male and female rats at 100, 300, and 1000 mg/kg/day, the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day, the highest dose level evaluated. No adverse signs of systemic toxicity or any signs of neurotoxicity were evident.
Executive summary:

The objective of the study was to evaluate the potential toxicity of the test article, Hatcol® 3346, after oral gavage administration to rats for 90 consecutive days.

 

The study was conducted in accordance with MPI Research Standard Operating Procedures (SOPs) and the protocol as approved by the Sponsor. SOP and protocol deviations were acknowledged by the Study Director and documented in the raw data. 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 OECD Guideline 408; the United States EPA, Office of Prevention, Pesticides, and Toxic Substances (OPPTS) 870.3100; and the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011.

 

Assessment of toxicity was based on mortality, cage side clinical observations, detailed clinical observations, neurobehavioral evaluations, locomotor activity, functional observational battery, body weight, food consumption, ophthalmoscopic examinations, and clinical and anatomic pathology.

 

There were no mortalities, test article-related clinical findings, ophthalmologic findings, or test article-related changes in body weight or food consumption for males or females at any dose level. No correlative/supportive test article-related effects were noted in the neurobehavioral, FOB or locomotor evaluations. Test article-related clinical pathology changes observed included: prolongations in activated partial thromboplastin time in males at ≥100 mg/kg/day; increases in neutrophils in males at 1000 mg/kg/day that lacked correlative findings in other study endpoints; decreases in red cell mass and increases in platelets in females at 1000 mg/kg/day; decreases in potassium and phosphorus and increases in total protein, albumin and globulin in females at 1000 mg/kg/day that lacked correlative findings in other study endpoints. There were no test article-related macroscopic or organ weight changes. Test article-related microscopic changes consisted of vacuolated macrophages in the lungs in males and females at all dose levels and follicular cell hypertrophy in the thyroid glands of males at 1000 mg/kg/day and in females at ≥300 mg/kg/day. These findings were not considered adverse based on the minimal to mild severity.

 

Under the conditions of the study, where Hatcol® 3346 was administered via oral gavage for 90 consecutive days to male and female rats at 100, 300, and 1000 mg/kg/day, the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day, the highest dose level evaluated. No adverse signs of systemic toxicity or any signs of neurotoxicity were evident.