4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Endpoint summary

Description of key information

Repeated Dose Oral Toxicity: 28 day NOAEL 40 mg/kg bw/day 
Repeated Dose Oral Toxicity: 90 day NOAEL 100 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 14, 2015 to March 22, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

On Day 27, a record starting the stirring of the test article was not documented. In the opinion of the Study Director, this deviation did not affect the quality or integrity of the study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

yes

Remarks:

On Day 27, a record starting the stirring of the test article was not documented. In the opinion of the Study Director, this deviation did not affect the quality or integrity of the study.

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Acquisition and Acclimation

A total of 46 male and 46 female experimentally naïve CD®[Crl:CD®(SD)] rats, approximately 6 weeks of age at receipt, were received from Charles River Laboratories, Raleigh, North Carolina on September 22, 2015. During the 14–day acclimation period, the animals were observed daily with respect to general health and any signs of disease. The animals were acclimated to the dosing procedure on two occasions the week prior to vehicle or test article administration via a sham dose of tap water at the same dose volume intended for use on study.

Randomization, Assignment to Study, and Maintenance
Using a standard, by weight, measured value randomization procedure, 40 male and 40 female animals (weighing 247 to 288 g and 180 to 218 g, respectively, at randomization were assigned to the control and treatment groups. 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, with the exception of two male and two female animals which were transferred to the stock 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.Upon arrival and throughout the course of the study, animals were same sex pair-housed in solid bottom cages with nonaromatic bedding in an environmentally controlled room. Animal enrichment was provided according to SOP. The FOB-designated animals were individually housed prior to testing. 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 68 to 79 °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 designated periods. 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 SOP. 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.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose (400cps) in deionized water,

Details on oral exposure:

Date Received: July 14, 2015

Received From: Sigma-Aldrich, Inc, Milwaukee, Wisconsin, United States

Amount Received: 6 containers with approximately 6,000 g

Label Identification: Methylcellulose-400cps

Batch Number: SLBM2910V

Physical Characteristics: Light, off-white powder

Retest Date: January 2018

Storage: Room temperature

TMC Number: 1505A5

Vehicle and Test Article Preparation Fresh vehicle, 0.5% methylcellulose (400cps) in deionized water, was prepared for use on study weekly and was stored at room temperature. On occasion, additional vehicle preparations were made during the course of the study.The test article, Naugard® 445, was used as received. No adjustment was made for purity when preparing the test article formulations. Formulations of the test article were prepared by mixing the appropriate amount of vehicle with the appropriate amount of test article at nominal concentrations of 1, 3 and 10 mg/mL. Formulations were prepared weekly and were stored refrigerated at 2 to 8 °C until acquired for dosing.

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 were collected using a positive displacement pipette, while the formulations were stirring and placed into 20 mL amber glass scintillation vials.

Analyses
All analytical work was conducted by MPI Research, using an analytical method developed and validated under MPI Research Study Number 1038-051. No deviations from the analytical method occurred during the course of this study.

ANALYTICAL SUMMARY

Objective: To determine the homogeneity and concentration of Naugard® 445 in dosing formulations.

Compliance: This nonclinical laboratory study phase was conducted in accordance with the United States Environmental Protection Agency (EPA), Toxic Substances Control Act (TSCA) Good Laboratory Practice (GLP) Regulations, 40 Code of Federal Regulations (CFR) Part 792, and the Organization for Economic Cooperation and Development (OECD) Principles on GLP [as revised in 1997; ENV/MC/CHEM(98)17].

Analytical Method Number and Title: 1038-051: Determination of Naugard® 445 in 0.5% Methylcellulose (400 cps) in Deionized Water Dose Formulations by HPLC-UV

Reference Standard: Naugard® 445 Powder

Batch Number: EL5G14H256

MPI Research Inventory ID: 150658 (SP5004118)

Storage: Room temperature

Correction Factor: None

Data Collection and Analysis Software:Empower™ 2: Build 2154ExyLIMS Version 3.0NextDocs® v6.1Protocol Familiarization System v1.1, v1.2

HPLC Conditions: Liquid chromatography system equipped with a Waters XBridge Phenyl column, 3.0 x 100 mm, 3.5 μm particle size, and a Waters XBridge Phenyl guard column, 3.0 x 20 mm, 3.5 μm particle size, with a gradient flow of water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.75 mL/minute.

Analysis Description: Prior to analysis, duplicate samples were diluted with methanol (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 280 nm.

Regression Type: Linear, unweighted

Study Sample Receipt(s): Number of Samples: 144

Date(s) of receipt by Analytical Department: October 5, 2015 to December 28, 2015

Study Sample Storage Conditions: Refrigerated (2 to 8°C), protected from light

Storage Stability: All samples were analyzed within the established storage stability determined under MPI Research Study Number 1038-051(1).

Analysis Period: October 5, 2015 to December 28, 2015

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 limit of quantitation (LOQ)
Assessments
Homogeneity: 1. Average concentration within ±15% of the nominal concentration
2. Precision ≤10% RSD
Concentration:
1. Average concentration within ±15% of the nominal concentration
2. Precision ≤10% RSD3. Vehicle (control) samples < effective limit of quantitation (ELOQ)

Conclusion: A total of 48 samples were analyzed for Naugard® 445 in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results.Deviation: Standard Operating Procedure (SOP) Deviation: An SOP deviation was acknowledged by the Study Director and documented in the raw data. In the opinion of the Study Director, the SOP deviation was not considered to have affected the quality or integrity of the study.

Duration of treatment / exposure:

The vehicle and test article were administered for 91 days during the study via oral gavage.

Frequency of treatment:

The vehicle and test article were administered daily during the study via oral gavage.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

See Study Design table in Any other information.

Control animals:

yes, concurrent vehicle

Details on study design:

Justification of Test System

The current state of scientific knowledge 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 the 90-day study.

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 previous acute oral study in rat and a 28-day oral repeated dose study in rat on file from Hameln rds a.s., Dept. of Biology, Horná 36, 900 01 Modra, Slovak Republic.

Positive control:

Postive control not require for this study.

Observations and examinations performed and frequency:

In-life Examinations
Cage side Observations
All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily.

Cage side Clinical Examinations
A cage side clinical examination of each animal was performed daily on days that detailed clinical observations/neurobehavioral evaluations were not conducted. 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.

Detailed Clinical Observations
A detailed clinical examination of each animal was performed prior to randomization and weekly during the study. The examinations conducted prior to randomization are not reported but are maintained in the study file. The observations included, but were not limited to changes in the skin, fur, eyes, mucous membranes, and occurrence of secretions and excretions. On occasion, clinical observations were recorded at unscheduled intervals.

Neurobehavioral Evaluations
Neurobehavioral evaluations were conducted weekly on each animal 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 (i.e., morning or afternoon), each week. Animals were observed one at a time and 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 to autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, and reactivity to handling, 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. The ranking scales provided below were used for assessment.

Home Cage Observations
The animals were observed in the home cage for level of activity.
0 – Quiet or asleep
1 – Alert with routine movement (eating, sniffing at from of cage or similar movement)
2 – Increased movement

Open Field Observations
The animals were removed from the home cage and placed in the corner of a standard open-field testing box with a nine square grid. The animals were observed for one minute, and the number of grids entered with both forefeet were counted and recorded. While in the grid box, the animal was observed and scored as follows:
Tremors-
0 - Not present
1 – Present
Convulsions-
0 - Not present
1 – Present
Stereotypy-
0 - Not present
1 - Constant sniffing and periodic licking on the wall or floor
2 – Constant licking on the wall or floor
3 – Constant licking and periodic biting/gnawing of the wall or floor
4 – Constant biting/gnawing of the wall or floor
5 – Biting/gnawing of the body
6 – Self-mutilation
Gait-
0 - Normal
1 - Abnormal

Bizarre Behavior
-0 - Not present
1 – Present

Auditory Stimulus-(A clicker was placed approximately 5 cm above the back of the animal and a click was sounded suddenly.)
0 - Responded
1 - No response
2 - Exaggerated response

Grip Response-
Prior to the return of the animal to the cage, the forefeet were placed on the front of the cage or on any surface where the animal was able to demonstrate the ability to grip.
0 – Gripped cage
1 – Did not grip cage

Veterinary Observations
On occasion, veterinary observations were conducted during the course of the study. All observations were recorded.

Functional Observational Battery
Functional Observational Battery (FOB) evaluations were conducted without knowledge on the part of the testers of the treatment groups on all designated animals during Week 13. 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.

Motor Activity
Motor activity evaluations were conducted on all designated animals during Week 13. Each animal was placed into the assigned Hamilton-Kinder enclosure for monitoring. The duration of monitoring was 30 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 at receipt, prior to randomization and weekly during the study. The body weights recorded at receipt are not reported but are maintained in the study file.Food ConsumptionFood consumption was measured and recorded weekly during the study.

Ophthalmoscopic Examinations
Ophthalmoscopic examinations were conducted on all animals pretest and within 2 days of the end of treatment by Joshua T. Bartoe, DVM, DACVO.Clinical Pathology

Clinical pathology evaluations were conducted on all animals prior to the terminal necropsy.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.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:

Postmortem Study EvaluationsPostmortem study evaluations were performed on all animals at the scheduled terminal necropsy.

Other examinations:

None specified in the study report.

Statistics:

Reported in table form - See Any other information

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical findings noted related to the test article. Any findings noted were typical for this age and strain of rats.
One male animal at 10 mg/kg/day (animal number 2004) was reported for hind limb impairment on Day 85. As this did not resolve on its own, the animal was administered meloxicam for 3 doses. This treatment was necessary to maintain animal welfare and did not affect the outcome of this study as the treatment was short in duration and resulted in no adverse findings.

Mortality:

no mortality observed

Description (incidence):

No mortalities occurred during the conduct of this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no body weight effects related to the test article.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test article-related effects on food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test article-related ophthalmoscopic findings.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test article-related effects on hematology endpoints in any treatment group at the terminal collection. Minimal statistically significant decreases in reticulocyte counts were present in females at 30 (-22%) and 100 mg/kg/day (-26%), relative to controls, that were not considered test article-related or biologically meaningful due to their small magnitude, lack of correlative changes in red cell mass, and considerable overlap of individual values with controls. All other fluctuations among individual and mean values were considered sporadic, consistent with biologic variation, and/or negligible in magnitude, and not related to test article administration.

Coagulation
There were no test article-related effects on coagulation times (i.e. APTT and prothrombin time) in any treatment group at the terminal collection. All fluctuations among individual and mean values were considered sporadic, consistent with biologic variation, and/or negligible in magnitude, and not related to test article administration.

Clinical Chemistry
There were no test article-related effects on clinical chemistry analytes in any treatment group at the terminal collection. A statistically significant minimal decrease in alkaline phosphatase activity (ALP; -26%) was present in males at 100 mg/kg/day, relative to controls, that was not considered test article-related or biological relevant given the small magnitude and direction of the change, and lack of correlative findings in other study endpoints. 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.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The weekly neurobehavioral assessments in the treated animals were consistent with the control animals.
There were no test article-related effects on any FOB parameter. A statistically significant increase in hind limb grip strength among females at 100 mg/kg/day was not considered test article-related due to the direction of the change.
There were no test article-related changes noted in the motor activity assessment. At 30 mg/kg/day, statistically significant increases in basic movement, rearing, and total distance were noted in males; however, these are not considered test article-related as there was not a dose response evident.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test article-related organ weight changes.
The following organ weights in females were statistically different from controls: kidney weights relative to body weights at 10 mg/kg/day, liver weights relative to body weights at 10 and 30 mg/kg/day, and absolute thymus weights at 10 mg/kg/day. These organ weight changes were considered the result of normal biological variation based on the lack of dose response and the minimal magnitude.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test article-related microscopic changes.
The microscopic findings noted in the study are commonly encountered in Sprague-Dawley rats and were considered incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No observations noted.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No observations noted.

Other effects:

no effects observed

Details on results:

Analysis of Dosing Formulations
The results indicate the formulations were homogenous (%RSD < 5) and accurately prepared (+ 11% from nominal concentrations).

In-life ExaminationsMortality
No mortalities occurred during the conduct of this study.

Cage side Clinical Examinations and Detailed Clinical Observations
There were no clinical findings noted related to the test article. Any findings noted were typical for this age and strain of rats.One male animal at 10 mg/kg/day (animal number 2004) was reported for hind limb impairment on Day 85. As this did not resolve on its own, the animal was administered meloxicam for 3 doses. This treatment was necessary to maintain animal welfare and did not affect the outcome of this study as the treatment was short in duration and resulted in no adverse findings.

Neurobehavioral Evaluations
The weekly neurobehavioral assessments in the treated animals were consistent with the control animals.

Functional Observational Battery Evaluations
There were no test article-related effects on any FOB parameter. A statistically significant increase in hind limb grip strength among females at 100 mg/kg/day was not considered test article-related due to the direction of the change.

Motor Activity
There were no test article-related changes noted in the motor activity assessment. At 30 mg/kg/day, statistically significant increases in basic movement, rearing, and total distance were noted in males; however, these are not considered test article-related as there was not a dose response evident.

Body Weights
There were no body weight effects related to the test article.

Food Consumption
There were no test article-related effects on food consumption.

Ophthalmoscopic Examinations
There were no test article-related ophthalmoscopic findings.

Clinical Pathology
Once daily oral administration at 10, 30 or 100 mg/kg/day to rats for up to 91 consecutive days was not associated with any effects on clinical pathology endpoints.

Hematology
There were no test article-related effects on hematology endpoints in any treatment group at the terminal collection. Minimal statistically significant decreases in reticulocyte counts were present in females at 30 (-22%) and 100 mg/kg/day (-26%), relative to controls, that were not considered test article-related or biologically meaningful due to their small magnitude, lack of correlative changes in red cell mass, and considerable overlap of individual values with controls. All other fluctuations among individual and mean values were considered sporadic, consistent with biologic variation, and/or negligible in magnitude, and not related to test article administration.

Coagulation
There were no test article-related effects on coagulation times (i.e. APTT and prothrombin time) in any treatment group at the terminal collection. All fluctuations among individual and mean values were considered sporadic, consistent with biologic variation, and/or negligible in magnitude, and not related to test article administration.

Clinical Chemistry
There were no test article-related effects on clinical chemistry analytes in any treatment group at the terminal collection. A statistically significant minimal decrease in alkaline phosphatase activity (ALP; -26%) was present in males at 100 mg/kg/day, relative to controls, that was not considered test article-related or biological relevant given the small magnitude and direction of the change, and lack of correlative findings in other study endpoints. 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 Evaluations
Macroscopic
There were no test article-related macroscopic changes.The macroscopic findings noted in the study are commonly encountered in Sprague-Dawley rats and were considered incidental.

Organ Weights
here were no test article-related organ weight changes.The following organ weights in females were statistically different from controls: kidney weights relative to body weights at 10 mg/kg/day, liver weights relative to body weights at 10 and 30 mg/kg/day, and absolute thymus weights at 10 mg/kg/day. These organ weight changes were considered the result of normal biological variation based on the lack of dose response and the minimal magnitude.

Microscopic
There were no test article-related microscopic changes.The microscopic findings noted in the study are commonly encountered in Sprague-Dawley rats and were considered incidental.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test article-related effects noted on any parameter examined.

Key result

Critical effects observed:

no

See attached background materials for the following tables:

Summary of Cage side Clinical Examinations - MALE

Summary of Cage side Clinical Examinations - FEMALE

Summary of Detailed Clinical Observations - MALE

Summary of Detailed Clinical Observations - FEMALE

Summary of Neurobehavioral Findings- MALE

Summary of Neurobehavioral Findings- FEMALE

Summary of Functional Observational Battery (Categorical Endpoints) – MALE

Summary of Functional Observational Battery (Continuous Endpoints) – MALE

Summary of Functional Observational Battery (Categorical Endpoints) – FEMALE

Summary of Functional Observational Battery (Continuous Endpoints) – FEMALE

Summary of Motor Activity – MALE

Basic Movement (count) - Week 13

Fine Movement (count) - Week 13

Rearing (count) - Week 13

Total Distance (cm) - Week 13

Summary of Motor Activity – FEMALE

Basic Movement (count) - Week 13

Fine Movement (count) - Week 13

Rearing (count) -Week 13

Total Distance (cm) - Week 13

Summary of Body Weight Values - MALE

Summary of Body Weight Values - FEMALE

Summary of Caged Food Consumption Values Per Animal - MALE

Summary of Caged Food Consumption Values Per Animal - FEMALE

Summary of Hematology Values - MALE

Summary of Hematology Values - FEMALE

Summary of Coagulation Values - MALE

Summary of Coagulation Values - FEMALE

Summary of Clinical Chemistry Values - MALE

Summary of Clinical Chemistry Values - FEMALE

Summary of Macroscopic Observations - MALE

Summary of Macroscopic Observations - FE  MALE

Summary of Organ Weight Values – MALE

Summary of Organ Weight Values – FEMALE

Summary of Microscopic Observations – MALE

Summary of Microscopic Observations – FEMALE

Conclusions:

Following 13 weeks of once daily oral gavage administration of Naugard® 445 at 0, 10, 30, or 100 mg/kg/day, the no observed adverse effect level (NOAEL) was 100 mg/kg/day, the highest dose level tested. There were no test article-related effects noted on any parameter examined.

Executive summary:

The objective of the study was to evaluate the potential toxicity of Naugard® 445 after oral gavage administration to rats for up to 91 days.

 

The study was based on the United States Environmental Protection Agency, Office of Prevention, Pesticides, and Toxic Substances, Guideline 870.3100, 90-Day oral toxicity in rodent, August 1998, the Organization for Economic Cooperation and Development (OECD) Guideline 408.

 

The study was conducted for the Chemtura Corporation to evaluate the potential toxicity of the test article, Naugard® 445, after oral gavage administration to rats for up to 91 days.

Animals were assigned to the study and were administered the vehicle or test article once daily for up to 91 days via oral gavage.

 

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

 

No test article-related effects were noted in any parameter examined. Therefore, the no observed adverse effect level (NOAEL) was 100 mg/kg/day, the highest dose level tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

K1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated Dose Oral Toxicity - 90 Day

This study was conducted for the Chemtura Corporation to evaluate the potential toxicity of the test article, Naugard® 445, after oral gavage administration to rats for up to 91 days.

Animals were assigned to the study as indicated below and were administered the vehicle or test article once daily for up to 91 days via oral gavage.

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

No test article-related effects were noted in any parameter examined. Therefore, the no observed adverse effect level (NOAEL) was 100 mg/kg/day, the highest dose level tested.

Repeated Dose Oral Toxicity - 28 day

The Wistar rats, 64 males and 64 females were divided into four groups. Three groups obtained the test article Dusantox 86 in graduated doses. The low dose level – 10mg/kg was estimated from available information about the test article. The high dose level – 80mg/kg, represents 8-fold of the low dose level. The medium dose level – 40mg/kg, represents 4-fold of the low dose level. Dusantox is not soluble in water, the olive oil was used as a vehicle. The test article was administered per os with help of a metal stomach tube, every day for 28 days. Control group obtained vehicle (olive oil). Doses were calculated on the current bodyweight. The animals were weighed once every two days. Fixed application volume 0.5mL/100g bodyweight was administered. Satellite animals from control and the highest dose groups were further observed for the period of 14 days after the last application of the test article for evaluation of reversibility or persistence of any toxic effects. During the study clinical observation, bodyweight and food consumption were recorded. Blood for hematological and clinical chemistry investigation was collected before application and after 28 days, in animals of satellite groups 14 days after the last application. After finishing of the test, the animals were sacrificed and moved to histological evaluation.

 

Consideration of the results of this study it is possible to state:

The test article Dusantox 86 did not cause treatment-related deaths of animals. 

The test article Dusantox 86 caused frequent incidence of smooth stools, decrease of creatinine and increase of total bilirubin, increase of triacylglycerols and induced retarded decrease of total cholesterol in males of dose group 80mg/kg. The test article Dusantox 86 caused increase of Quick test values in females of dose group 80mg/kg. The test article Dusantox 86 caused increase of ALP in males and females in dose group 80mg/kg. The test article Dusantox 86 produced moderate degenerative changes “nephrosis” in proximal tubules females’ kidneys of all dose groups.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study performed to OECD guideline No. 408.

Justification for classification or non-classification

The above studies have been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies are conducted to GLP and in accordance with an appropriate OECD Guideline. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

 

Review of the effects noted in the 28 -day study are read in conjunction with the toxicokinetics report on the substance.  The result of this is as follows:

 

• The kidney effects noted in the females only are not dose dependant.

• The report indicates that these are degenerative effects, not necrotic effects. As such the report states that these are “probably reversible”.

• The satellite group females appear to be recovering in that the incidence is significantly lower at 14 days and adding further evidence of reversibility.

• The toxicokinetics study indicates that the substance is not absorbed by the kidneys.

 

As a result of this, it is proposed that the effects noted are due to adaptive changes, rather than actual toxicity, particularly as the effects noted do not seem to be dose dependant. The effects may also be specific to the rat strain (Wistar) used in the study. It is well-known from the literature (Barthold, 1979; Gray et al., 1982) that chronic progressive nephropathy (CPN) is a common disease occurring spontaneously in various strains of laboratory rats. The lesion affects female rats earlier and more severely than  male animals which may explain the sex-specificity of the findings in this study. The syndrome is not inflammatory and its basic etiology and early pathogenesis are not completely understood (Barthold, 1979). Gray (1986) mentions that in long-term toxicity studies with certain compounds, a dose-related acceleration of CPN may occur. The gross appearance of the kidneys of rats with CPN was described by Gray (1986) to be swollen and pale with irregular, indented surfaces.

This opinion is reinforced by the text of the CLP Regulation (EC No 1272/2008), specifically section 3.9.2.9.9 which states that:

 

“Thus it is feasible that a specific profile of toxicity occurs in repeat-dose animal studies at a dose/concentration below the guidance value, such as < 100 mg/kg bw/day by the oral route, however the nature of the effect, such as nephrotoxicity seen only in male rats of a particular strain known to be susceptible to this effect may result in the decision not to classify. “

 

The effects noted appear to be specific to females, but could be considered to be applicable to the Wistar strain, as this this is documented, albeit in males rather than females.

 

References: Barthold, S.W. Chronic Progressive Nephropathy in Aging Rats. Toxicol. Pathol. 7, 1-6, 1979 Gray, J.E. et al. Early Light Microscopic Changes of Chronic Progressive Nephrosis in Several Strains of Aging Laboratory Rats, J. Gerontol, 37, 142-150, 1982  Gray, J.E. Chronic Progressive Nephrosis, Rat, in: Jones, T.C. et al. (eds.): Urinary System, Monographs on Pathology of Laboratory Animals, Springer, Berlin, 1986, pp. 174-179

 

Therefore classification and labelling is not attributable to these effects.

 

The results of the OECD 408 90 -day study confirm the above opinion that the effects noted were species specific, as similar results are not noted within this sub-chronic test that utilised the Sprague-Dawley strain of rats.

 

Discussion on classification:

 

The classification requirements for this are detailed in CLP as follows:

 

3.9.2.9.7. Classification in Category 2 is applicable, when significant toxic effects observed in a 90-day repeated-dose study conducted in experimental animals are seen to occur within the guidance value ranges as indicated in Table 3.9.3.. These effects were not noted in the 90 -day study conducted.

  

The above results are considered to trigger no classification under the CLP Regulation (EC No 1272/2008)