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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:
30 January 2015 to 05 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This testing was performed for the purpose of chemical notification in China. Therefore no testing proposal was included.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Details on test material:
EnvaMul200
Lot# D-0067
Expiry date: 11-2-2016

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
appropriate for repeated dose studies
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston, NY
- Females nulliparous and non-pregnant: no data
- Age at study initiation: ca 51 days
- Weight at study initiation: males: 221-254 g, females 159-196 g
- Fasting period before study: no
- Housing: 2-3/sex/cage in solid bottom cages containing ground corncob bedding material
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 ad libitum
- Water: reverse osmosis treated water ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: no contaminants present

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3°C to 22.2°C
- Humidity (%): 25.8% to 61.6%
- Air changes (per hr): at least 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 February 2015 To: 18/19 May 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle was dispensed approximately weekly for administration to the control group and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light. The vehicle was mixed throughout the sampling and dose administration procedures. The formulations were stored protected from light (at 18-24 °C).


VEHICLE
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL - Amount of vehicle (if gavage): 10 mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before study start and in week 4, 8 and 12 from formulation preparations. Samples were analysed for accuracy, homgeneity (start from all concentrations) and stability (before study start reanalysed one day thereafter) in duplicate.
Samples were prepared in ethanol and vortexed before analysis by HPLC-UV

Instrument: Agilent 1100 liquid chromatograph equipped with a variable
wavelength detector, autosampler, and Dionex Chromeleon
software version 6.8, or equivalent system
Column: Phenomenex Luna C8(2), 150 mm × 4.6 mm, 5.0-µm particle-size; Guard Column/Cartridge: Phenomenex Security Guard C18 4.0 × 2.0mm
Mobile Phase: A: 0.1% TFA in DI water
B: 0.1% TFA in ACN
Flow Rate: 1.00 mL/minute
Gradient: Time(minutes) A(%) B(%)
0 90 10
2.0 90 10
7.0 10 90
9.0 10 90
9.1 90 10
15.0 90 10
Column Temperature: 40°C
Autosampler Temperature: Ambient
Detector: UV at 230 nm
Injection Volume: 30 µL

Calibration range 8 - 40 µg/mL (no info on linearity)
QC samples: at 100 mg/mL in ethanol (mixed and sonicated): 94.6-98.8 % of nominal
Stability (17 days): reported to be stable
Homogeneity: at 10 mg/mL 99.2 ± 1.1%, at 30 mg/mL 97.8 ± 1.7%, at 100 mg/mL 97.8 ± 6.3%
Duration of treatment / exposure:
90-91 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
measured: 94.8-115% of nominal
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
measured: 96.1-114% of nominal
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
measured: 91.6-114% of nominal
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 28 day study with a NOAEL of 1000 mg/kg bw

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality/during and 1-2 hours after dosing for clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (starting with randamization)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOODCONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatization and in week 12
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of necropsy
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: all
- Parameters: Total leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC ABSOLUTE), Mean platelet volume (MPV),
Differential leukocyte count: Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC)), Red cell distribution width (RDW), Hemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day of necropsy
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: all
- Parameters: Albumin, Total protein, Globulin, Albumin/globulin ratio (A/G Ratio) , Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Sorbitol dehydrogenase (SDH), Triglycerides (Triglyceride), Creatine kinase (CK), Bile Acids (Bile acid)
- Appearance: degree of hemolysis, lipemia, and icterus

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: on day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes overnight
- Parameters: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Color (COL), Clarity (CLA), Protein (PRO), Glucose (GLU), Ketones (KET), Bilirubin (BIL), Occult blood (BLD)
Leukocytes (LEU), Nitrites (NIT, Microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: in week 11/12
- Dose groups that were examined: all
- Battery of functions tested:
HOME CAGE OBSERVATIONS: Posture, Convulsions/tremors, Biting, Palpebral (eyelid) fissure, Feces consistency
HANDLING OBSERVATIONS: Ease of removal from cage, Ease of handling animal in hand, Lacrimation/chromodacryorrhea, Salivation, Piloerection, Fur appearance, Palpebral closure, Respiratory rate/character, Red/crusty deposits, Mucous membranes/eye/skin color, Eye prominence, Muscle tone
OPEN FIELD OBSERVATIONS (over a 2-minute observation period): Time to first step (seconds), Mobility. Gait, Convulsions/tremors, Gait score, Arousal, Bizarre/stereotypic behaviour, Rearing, Backing, Grooming, Urination/defecation
SENSORY OBSERVATIONS: Approach response, Touch response, Startle response, Tail pinch response, Olfactory orientation, Pupil response, Eyeblink response, Forelimb extension, Hindlimb extension, Air righting reflex
NEUROMUSCULAR OBSERVATIONS: Hindlimb extensor strength, Grip strength-hind and forelimb, Rotarod performance, Hindlimb foot splay
MOTORACTIVITY: ambulatory and total motor activity over six, 10-minute subintervals

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes on all animals
Adrenals, Aorta, Bone with marrow (Femur and Sternum), Bone marrow smear (from femur), Brain, Cervix, Epididymides, Eyes with optic nerves, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys, Larynx, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph nodes, Axillary (2), Mandibular (2), Mesenteric, Ovaries with oviducts, Pancreas, Peripheral nerve (sciatic), Peyer’s patches, Pharynx, Pituitary, Prostate, Salivary glands (mandibular), Seminal vesicles, Skeletal muscle (rectus femoris), Skin (with mammary gland), Spinal cord (cervical, thoracic, lumbar), Spleen, Testes, Thymus, Thyroid (with parathyroids), Tongue, Trachea, Urinary bladder, Uterus, Vagina, Gross lesions

HISTOPATHOLOGY: Yes from all control and high dose animals
Adrenals, Aorta, Bone with marrow (Femur and Sternum), Bone marrow smear (from femur), Brain, Cervix, Epididymides, Eyes with optic nerves, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys, Larynx, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph nodes, Axillary (2), Mandibular (2), Mesenteric, Ovaries with oviducts, Pancreas, Peripheral nerve (sciatic), Peyer’s patches, Pharynx, Pituitary, Prostate, Salivary glands (mandibular), Seminal vesicles, Skeletal muscle (rectus femoris), Skin (with mammary gland), Spinal cord (cervical, thoracic, lumbar), Spleen, Testes, Thymus, Thyroid (with parathyroids), Tongue, Trachea, Urinary bladder, Uterus, Vagina,

From all animals :Gross lesions

ORGAN WEIGHTS: Yes from all animals
Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Pituitary, Prostate with seminal vesicles, Spleen, Testes, Thymus, Thyroid with parathyroids, Uterus
Statistics:
Body weight, body weight change, food consumption, continuous FOB, clinical pathology, and organ weight data were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used. Functional ob servational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test

Motor activity with RANOVA, pairwise comparisons were made for each individual test item-treated group with the control group

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item-related, nonadverse clinical observations of red material around the mouth were
noted in all test-item treated male and female groups at 1-2 hours following dose administration.
This observation was first noted beginning on study day 10 and was generally observed
throughout the remainder of the study period. Furthermore, this observation was generally noted
more frequently in males compared to females, was noted with greater incidence in the
1000 mg/kg/day group compared to the 100 and 300 mg/kg/day groups, and was considered to
be attributed to the color of the test item dosing formulations.
All other clinical findings in the test item-treated groups were noted with similar incidence in the
control group, were limited to single animals, were not noted in a dose-related manner, and/or
were common findings for laboratory rats of this age and strain
see attached document for detailed tables
Mortality:
no mortality observed
Description (incidence):
No mortality in any of the dose groups or controls
see attached document for detailed tables
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: 1000 mg/kg bw decreased body weight gain 12% compared to controls over the test period (statistically significant during week 4-5 and 11 -12)
Body weights were unaffected by test item administration. Statistically significant lower mean
body weight gains were occasionally noted in the 300 and 1000 mg/kg/day group males; these
transient differences in body weight gain were attributed to biological variability and were not
considered related to test item administration.
see attached document for detailed tables
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related effects (occasionally slightly higher at 100 and 1000 mg/kg bw)
Food consumption was unaffected by test item administration. Statistically significant higher
food consumption values were occasionally noted in the 100 and 1000 mg/kg/day group females;
these transient differences in food consumption were attributed to biological variability and were
not considered related to test item administration.
See attached document for detailed tables
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmic lesions indicative of toxicity were observed in any of the test item-treated groups.
All findings observed were typical in prevalence and appearance for laboratory rats of this age
and strain.
see attached document for detailed tables
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males at 1000 mg/kg bw: sign increased APTT (+25.5%) and sign in creased PT (+ 26.4%)
Males at 300 mg/kg bw: sign increased PT (+15.3%)
Females: at 100, 300 and 1000 mg/kg bw: sign decreased number of eosinophils no dose relationship and within historical controls)
Females at 1000 mg/kg bw: sign decreased Hb (within historical controls)

Test item-related, nonadverse coagulation parameter alterations included higher prothrombin
time (PT) and activated thromboplastin time (APTT) values in the males, as described below.
Higher mean PT values were noted in the 300 and 1000 mg/kg/day group males ( 15.3% and
26.4%, respectively) at study week 12/13. A similar change was not observed in the test
item-treated female groups.
Higher mean APTT was noted in the 1000 mg/kg/day group males at study week 12/ 13 (25.5%).
A similar change was not observed in the test item-treated female groups.
There were no test item-related effects on hematology parameters and no additional test
item-related effects on coagulation parameters. However, some statistically significant
differences were observed when the control and test item -treated groups were compared.
Specifically, a lower mean hemoglobin value was noted in the 1000 mg/kg/day group females;
however, this change was of minimal magnitude and all group means were within the WIL
Research historical control database range of study means (version 3.6). A lower mean PT value
was noted in the 1000 mg/kg/day group females; however, this involved a change in a direction
of limited toxicological importance. Lower mean absolute eosinophil counts were noted in the
100, 300, and 1000 mg/kg/day group females; however, these changes did not show a clear
dose-related response and all group mean values were within the WIL Research historical control
database range of study means. Thus, these changes were not considered to be test item-related.
Statistically significant findings that involved percentage leukocyte differential counts
(specifically, percentage eosinophil counts in the 100 and 300 mg/kg/day group females) were
not itemized above, and were not considered toxicologically important because absolute cell
counts are more relevant for interpretative purposes.
see attached document for detailed tables
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males at 1000 ng/kg bw: sign increased ALP (+ 77%) and sign increased bilirubin (+ 400%)
Females at 1000 mg/kg bw: increased creatinine and cholesterol, decreased chloride values (within historical controls)

Test item-related, nonadverse serum chemistry alterations were limited to higher alkaline
phosphatase (ALP) value in the males, as described below.
A higher mean ALP value was noted in the 1000 mg/kg/day group males at s tudy week 12/13
(77.3%). A similar change was not observed in the test item-treated female groups.
There were no other test item-related effects on serum chemistry parameters. However, some
statistically significant differences were observed when the control and test item-treated groups
were compared. Specifically, a higher mean total bilirubin value was noted in the
1000 mg/kg/day group males; however, this change was of minimal magnitude and only
2 individual animal values were above the range of concurrent control group values. Higher
mean creatinine, higher mean cholesterol, and lower mean chloride values were noted in the
1000 mg/kg/day group females; however, these changes were of minimal magnitude and the
group means and all individual animal values in this group were within the WIL Research
historical control database range of study means and reference range (version 3.6), respectively.
A lower mean glucose value was noted in the 300 mg/kg/day group males and a higher mean
triglyceride value was noted in the 100 mg/kg/day group males; however, these changes did not
show a dose-related response. Thus, these changes were not considered to be test item-related.
see attached document for detailed tables
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Females at 300 and 1000 mg/kg bw: sign decreased total urine volume (no dose respone and within historical control values

There were no test item-related alterations in urinalysis parameters.
However, some statistically significant differences were observed when the control and test
item-treated groups were compared. Specifically, lower mean total urine volume values were
noted in the 300 and 1000 mg/kg/day group females at study week 13; however, all individual
animal values in these groups were within the range of concurrent control group values and the
WIL Research historical control database reference range (version 3.6). Thus, these changes
were not considered to be test item-related.
See attached document for detailed tables
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
HOME CAGE OBSERVATIONS, HANDLING OBSERVATIONS, OPEN FIELD OBSERVATIONS, SENSORY OBSERVATIONS AND NEUROMUSCULAR OBSERVATIONS: no treatment related effects

MOTORACTIVITY:No treatment related effects, no remarkable shifts in the pattern of habituation occurred in any of the groups

HOME CAGE OBSERVATIONS
Home cage observations were unaffected by test item administration. There were no statistically
significant differences when the test item-treated males and females were compared to the
control group at the study week 11/12 evaluation.
HANDLING OBSERVATIONS
Handling observations were unaffected by test item administration. There were no statistically
significant differences when the test item-treated males and females were compared to the
control group at the study week 11/12 evaluation.
OPEN FIELD OBSERVATIONS
Open field observations were unaffected by test item administration. There were no statistically
significant differences when the test item-treated males and females were compared to the
control group at the study week 11/12 evaluation.
SENSORY OBSERVATIONS
Sensory observations were unaffected by test item administration. There were no statistically
significant differences when the test item-treated males and females were compared to the
control group at the study week 11/12 evaluation.
NEUROMUSCULAR OBSERVATIONS
Neuromuscular observations were unaffected by test item administration. There were no
statistically significant differences when the test item-treated males and females were compared
to the control group at the study week 11/12 evaluation.
PHYSIOLOGICAL OBSERVATIONS
Physiological observations were unaffected by test item administration. There were no
statistically significant differences when the test item-treated males and females were compared
to the control group at the study week 11/12 evaluation.

MOTOR ACTIVITY
Motor activity patterns (total and ambulatory activity counts) were unaffected by test item
administration. There were no statistically significant changes in the test item-treated male and female groups when compared to the control group at the study week 11/12 evaluation. Values
obtained from the 6 epochs evaluated (0 -10 minutes, 11-20 minutes, 21-30 minutes,
31-40 minutes, 41-50 minutes, and 51-60 minutes) and the overall 60-minute test session were
comparable to the concurrent control values. No remarkable shifts in the pattern of habituation
occurred in any of the test item-treated groups when the animals were evaluated during study
week 11/12.
see attached document for detailed tables
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males at 1000 mg/kg bw: sign increased rel kidney weight (+10.2%) and abs/rel liver weight (+6.7/17 %); sign increased rel spleen weight (+24%); sign decreased abs prostate weight (-15.6%)
Males at 100 and 300 mg/kg bw: sign increased abs/rel spleen weight (no dose response, mainly within historical controls)
Females at 1000 mg/kg bw: sign increased abs/rel liver weight (+36.5/34.5 %); sign increased abs/rel spleen weight (+18.0/17.1%)
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related changes
Test item-related, non adverse organ weight differences were limited to higher liver weight in the
females, as described below.
Higher mean liver weight (absolute: 36.5%; relative to final body weight: 34.5%; and relative to
brain weight: 37.1%) were noted in the 1000 mg/kg/day group females at the scheduled
necropsy.
There were no other test item-related effects on organ weights. However, some statistically
significant differences were observed when the control and test item-treated groups were
compared. Specifically, higher mean kidneys weight relative to final body weight, higher mean
liver weight relative to final body weight, lower mean absolute seminal vesicles /prostate weight,
and higher mean spleen weight relative to final body weight were noted in the 1000 mg/kg/day
group males; however, these changes were due to a lower final body weight in this group.
Higher mean spleen weight (absolute, relative to final body weight, and relative to brain weight)
was noted in the 100 mg/kg/day group males; however, this change did not show a dose -related
response. Higher mean spleen weight (absolute, relative to final body weight, and relative to
brain weight) was noted i n the 1000 mg/kg/day group females; however, this change was of
minimal magnitude with only 2 individual animal values slightly above the WIL Research
historical control database observed value range. Thus, these changes were not considered to be
test item-related.
see attached document for detailed tables
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related changes
There were no test item-related histologic changes.
All histologic changes were considered to be incidental findings or related to some aspect of
experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
see attached document for detailed tables
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
1 female at 100 mg/kg bw had a mammary gland adenoma/carcinoma
Other effects:
no effects observed
Details on results:
The decreased body weigh gain in males at 1000 mg/kg was attributed to biologial variability
Effects on liver (and the increased ALP), kidney and spleen weights were not accompanied by any macroscopic or histopathological changes and were therefore considered to be non-adverse.
The increases in PT and ATTP are without any histopathological changes and therefore considered to be non-adverse.

data on historical controls were included for behavioral effects only.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no biologically relevant effects observed

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

During the last week of treatment, it could not be excluded that the dosage has been slightly higher than in the rest of the treatment period. This was due to the use of a new container of the substance where the analytical determinations showed concentrations to be 157 -161% of nominal at 100, 300 and 1000 mg/kg bw. As there was no change in clinical symptoms during the last study week and the cocnetrations used before were confirmed by analytical determination, this has not affected the study outcome.

Dose (mg/kg bw)

0

 

100

 

300

 

1000

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Mortality

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

No

Clinical signs (max no affected)

-hair loss

-wet material nose/forelimbs

 

 

1

0

 

 

2

0

 

 

1

1

 

 

1

5

 

 

2

2

 

 

0

10

 

 

2

2

 

 

2

10

 

No

incidental findings

Body weight ( mean gain wk 0-13 (g))

292

104

317

107

285

103

257

115

No

Food consumption

 

 

 

 

wk 9-10

 

 

wk 7-8, 9-10, 12-13

No

transient

Detailed clinical observations*

NTRE

No

Behavioral effects*

NTRE

No

Motoractivity (total movements 0-60 min)

2195

2321

2489

3320

2566

2754

2035

2275

No

Haematology

 

 

 

 

EOS

(-47%)

↑PT (15%)

EOS (-58%)

↑PT/APTT (24-26%)

HGB (-9%)

PT (-6%)

EOS (-53%)

No

PT non-adverse

HGB +EOS within historical control#

Clinical biochemistry

 

 

 

 

 

 

Total bilirubin (400%)

↑ALP (77%)

↑creatinine (15%)

↓Chloride (1.9%)

No

incidental or within historical control#

Urinalysis

 

 

 

Total volume

(-59%)

 

Total volume

 (-65%)

 

Total volume

 (-48%)

No

within historical control#

Ophthalmoscopy

NTRE

No

Organ weights

 

 

 

 

spleen (abs/rel 14-20%)

 

kidneys (rel 10%)

liver (rel 17%)

prostate

(abs -15%)

spleen (abs 15%)

liver (abs/rel 35-37%)

spleen (abs/rel 17-20%)

 

No

Spleen effects incidental

Liver effects no microscopic findings

Marcoscopy

NTRE

No

incidental findings

Histopathology

NTRE

No

*HOME CAGE OBSERVATIONS, HANDLING OBSERVATIONS, OPEN FIELD OBSERVATIONS (over a 2-minute observation period,

**SENSORY OBSERVATIONS and NEUROMUSCULAR OBSERVATIONS

# historical control data not provided in the study report

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

 

Applicant's summary and conclusion

Conclusions:
The NOAEL is 1000 mg/kg bw based on the absence of adverse effects
Executive summary:

The substance (in corn oil) was administered orally by gavage once daily for a minimum of 90 consecutive days to 3 toxicology groups (Groups 2-4) of Crl:CD(SD) rats.  Dosage levels were 100, 300, and 1000 mg/kg/day for Groups 2, 3, and 4, respectively.  A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 10 mL/kg for all groups. Each group consisted of

10 animals/sex.  Following a minimum of 90 days of dose administration, all animals were euthanized.

All animals were observed twice daily for mortality and moribundity.  Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body weights were recorded weekly (± 2 days).  Cage food weights were recorded once weekly (± 2 days) beginning following randomization. Functional observational battery (FOB) and motor activity data were recorded for all animals during study week 11/12.  Ophthalmic examinations were performed during study weeks -2 and 12.  Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals on the day of the scheduled necropsy (study week 12/13). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals in the control and 1000 mg/kg/day groups.  Gross lesions were examined from all animals.  

All animals survived to the scheduled necropsy. Bodyweight gain in males at 1000 mg/kg bw was slightly decreased. There were no substance-related effects on food consumption.  There were no substance-related effects noted during the functional observational battery and locomotor assessments.  There were no substance-related ophthalmic,urinalysis, macroscopic, or microscopic findings.

Substance-related, nonadverse clinical observations noted in all test item-treated male and female groups included red material around the mouth at 1-2 hours following dose administration.

Substance-related, alterations in coagulation parameters included nonadverse higher mean prothrombin time values in the 300 and 1000 mg/kg/day group males and a higher activated thromboplastin time values in the 1000 mg/kg/day group males.  Substance-related alterations in serum chemistry parameters included a nonadverse higher alkaline phosphatase value in the 1000 mg/kg/day group males.  

Substance-related alterations in organ weights included nonadverse effects on liver, kidney and spleen weight, As there were no histologic correlates associated with these alterations, they were not considered to be adverse.

Based on the results of this study, oral administration of the substance to Crl:CD(SD) rats for a minimum of 90 consecutive days resulted in a no-observed-adverse-effect level (NOAEL) of

1000 mg/kg/day, the highest dosage level tested.