Registration Dossier

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Diss Factsheets

Administrative data

Description of key information

 NOAEL 1000 mg/kg bw/day based on read across to structural analogue.

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Mar 2022 to 06 Jul 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guideline 422. Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
No further details specified in the study report.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Not specified in the study report
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species: Rat.
Strain: Crl: WI(Han).
Condition: Outbred, SPF-Quality.
Source of Test System: Charles River Deutschland, Sulzfeld, Germany.
Number of Males: 40
Number of Females: 48 (nulliparous and non-pregnant).
Age at Initiation of Dosing: Males 10 – 12 weeks old; Females 11 – 13 weeks old
Body Weight Range at Initiation of Dosing: 271 – 316 g males; 183 – 242 g females
Number of Acclimation Days: 8
Number of Treatment Days Males: 29; Females: 42 – 64

Animal Identification
Method: Each animal will be identified using a subcutaneously implanted electronic identification chip that is implanted prior to start of the pretest period (females) or treatment period (males).
Prior to start of the pretest period, reserve females will be numbered R1 through R8 at random by indelible marker. Any reserve female replacing an allocated female prior to treatment will receive
identification by chip.
Further identification marks may be applicable (e.g. tail mark with indelible ink), to be documented in the study file.

Environmental Acclimation
The animals will be allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days prior to start of the pretest period (females) or at least 5 days before the commencement of dosing (males).

Selection, Assignment, Replacement, and Disposition of Animals
A total of 40 females will be selected at randomization before initiation of the pretest phase.
Each selected female classified as not having regular estrous cycles during the pretest phase will be replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles will continue in the study. The supernumerary females will then be removed from the study, and their estrous cycle results will be kept in the raw data but will not be reported.
Animals will be randomly assigned to groups at arrival. Males and females will be randomized separately. Animals in poor health will not be assigned to groups.
At least upon receipt of the animals, a health inspection will be performed and any assigned animals considered unsuitable for use in the study will be replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
After initiation of dosing, study animals may be replaced during the replacement period with alternate animals in the event of accidental injury, non-test item-related health issues, or similar circumstances. The alternate animals may be used as replacements on the study within 1 to 3 days.

HUSBANDRY
Housing
Caging: On arrival and during the pretest (females only) and pre-mating period, animals will be group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
During locomotor activity monitoring, F0-animals will be housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Cages containing sterilized wooden fibers as bedding material
(Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
These housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the
study records. Cages will be arranged on the racks according to a Latin-square model.
Animals will be separated during designated procedures/activities.

Cage Identification: Color-coded cage card indicating at least Test Facility Study No., group, animal identification number.

Animal Enrichment
Animals will be socially housed for psychological/environmental enrichment and will be provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants that would interfere with the objectives of the study.

Environmental Conditions
The actual daily mean temperature during the study period was 19 to 21C with an actual daily mean
relative humidity of 35 to 67%. The values that were outside the targeted mean humidity range occurred on two days with a minimum of 35% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study
Light Cycle: 12-hours light and 12-hours dark (may be interrupted for designated
procedures).
Ventilation: At least 10 air changes per hour.

Food
Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
Type: Pellets (alternate diet may be provided on individual animal basis as warranted as approved by the Study Director).
Frequency: Ad libitum, except during designated procedures. During motor activity measurements, animals will not have access to food for a maximum of 2 hours.
Analysis: Results of analysis for nutritional components and environmental contaminants are provided by the Supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Type: Municipal tap water.
Frequency/Ration: Freely available to each animal via water bottles. During motor activity measurements, animals will not have access to water for a maximum of 2 hours.
Analysis: Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Details on oral exposure:
Preparation of Formulations
Dose formulations will be divided into aliquots where required to allow to be dispensed on each dosing occasion.
The dosing formulations will be removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations will be kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle will be continuously stirred until and during dosing.
Adjustments will be made for specific gravity of the vehicle and test item. No correction will be made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses will be performed using a validated analytical procedure.
Concentration and Homogeneity Analysis
Storage Conditions: Temperature set to maintain 18-22°C.
Acceptance Criteria: For concentration, mean sample concentration results within or equal to ± 10% for solutions and ± 15% for suspensions of theoretical concentration.
For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group.

Stability Analysis
Stability analyses will be performed, prior to the first dosing, in conjunction with the method development and validation study to demonstrate if the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.
Females: 7 days a week for at least 14 days prior to mating up to and including the day before scheduled necropsy.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 per dose group (10 male/10 female)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels for the Main study were based on the results of the Dose Range Finding Study. Up to 1000 mg/kg/day, no toxicity was observed, and this dose level was therefore selected being the high dose for this Main study.
Positive control:
No required for this study.
Observations and examinations performed and frequency:
Mortality: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency will be at least once daily.
Animals will be observed within their cage unless necessary for identification or confirmation of possible findings.

Clinical Observations: At least once daily, up to the day prior to necropsy. Animals will be observed for specific clinical signs. The time of onset, grade and duration of any observed signs will be recorded.
Signs will be graded for severity and the maximum grade will be predefined at 1, 2, 3 or 4.
Grades will be coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (i.e. maximum grade 1) will be scored. In the data tables, the scored grades will be reported, as well as the percentage of animals affected in summary tables.

Arena Observations: Once before the first administration of the test item and weekly during the Treatment Period.
Animals will be observed for clinical signs outside the home cage in a standard arena after dosing.
The time of onset, grade and duration of any observed signs will be recorded.

Individual Body Weights: On Day 1 of treatment (prior to dosing) and weekly thereafter.
In order to monitor the health status animals may be weighed more often.

Food Consumption: Weekly, except for males and females which are housed together for mating
and for females without evidence of mating.
Quantitatively measured per cage.

Water Consumption: Regular basis throughout the study.
Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

Functional Tests – F0-Generation
Frequency: Once during the Treatment Period. The 5 selected males per group will be tested once during Week 4 of treatment and the 5 selected females per group will be tested once during the last week of lactation (i.e. PND 6-13). These tests will be performed after clinical observations (including arena observation, if applicable).
Procedure: The following tests will be performed:
• hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength will be recorded as the mean of three measurements, using a grip strength meter (Series M4- 10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• locomotor activity (recording period: 1 hour under normal laboratory light conditions) will be tested using the Kinder Scientific Motor Monitor System LLC, Poway, USA. Total movements and ambulations will be reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

Clinical Pathology

Hematology
Hematology Parameters
White blood cells (WBC), Reticulocytes (absolute), Neutrophils (absolute), Red blood cell distribution (RDW), Lymphocytes (absolute), Hemoglobin, Monocytes (absolute), Hematocrit, Eosinophils (absolute), Mean corpuscular volume (MCV), Basophils (absolute), Mean corpuscular hemoglobin (MCH), Large unstained cells (LUC) (absolute), Red blood cells (RBC), Mean corpuscular hemoglobin concentration (MCHC), Platelets
A blood smear will be prepared from each hematology sample. Blood smears are labelled, stained, and stored. These smears will not be examined, but may be evaluated when required to confirm analyzer results.

Coagulation
Coagulation Parameters
Prothrombin Time (PT), Activated partial thromboplastin time (APTT)

Clinical Chemistry
Clinical Chemistry Parameters
Alanine aminotransferase (ALT), Creatinine, Aspartate aminotransferase (AST), Glucose, Alkaline phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total bilirubin, Chloride, Bile acids, Calcium, Urea, Inorganic phosphate (Inorg. Phos)

Thyroid Hormone
Thyroid Hormone Parameters
Thyroxine (T4), Thyroid-stimulating hormone (TSH; only if required)

After clotting and centrifugation, serum will be used as listed below.
F0-males: 150 μL serum for measurement of total T4 and the remaining volume of serum for possible future measurement of thyroid-stimulating hormone (TSH) (added by study plan amendment if applicable).
F0-females: The serum will be used for possible future measurement of total T4 and TSH (added by study plan amendment if applicable).
Serum samples retained for possible future analysis will be maintained by the Test Facility in the freezer (≤ -75°C). Under these storage conditions, samples will be stable for 6 months.
Any remaining sample will be discarded prior to finalization of the report.
Sacrifice and pathology:
Unscheduled Deaths/Euthanasia
If an animal dies on study, a necropsy will be conducted and specified tissues will be saved, but not weighed. If necessary, the animal will be refrigerated to minimize autolysis.
Animals may be euthanized for humane reasons as per Test Facility SOPs. These animals will be deeply anesthetized using isoflurane and subsequently exsanguinated. They will undergo necropsy, and specified tissues will be retained, but not weighed.

Scheduled Euthanasia
Animals surviving until scheduled euthanasia will have a terminal body weight recorded and will be deeply anesthetized using isoflurane and subsequently exsanguinated.

Scheduled necropsies are summarized below:
Males (which sire or fail to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which deliver: PND 14-16.
Females which fail to deliver: With evidence of mating: Post-coitum Days 25-27.
Without evidence of mating: Approximately 24-26 days after the last day of the mating period.
All males surviving to scheduled necropsy will be fasted overnight with a maximum of 24 hours before necropsy. Water will be available. F0-females will not be fasted overnight.

Necropsy
All animals will be subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites will be recorded for all paired females.

Organ weights
The organs will be weighed at necropsy for all scheduled euthanasia animals and females with total litter loss. Organ weights will not be recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs will be weighed together. Organ weights as a percent of body weight (using the terminal body weight) will be calculated.

Tissue Collection and Preservation
Representative samples of the tissues will be collected from all animals and preserved in 10% neutral buffered formalin or modified Davidson's solution as detailed in Test Facility SOPs. Additional tissue samples may be collected to elucidate abnormal findings.

HISTOLOGY AND MICROSCOPIC EVALUATION
Histology
Tissues will be embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin.

Microscopic Evaluation
Tissues will be evaluated histopathologically. Target tissues identified by the study pathologist
during microscopic evaluation will be communicated to the Study Director; tissues will be
evaluated and reported.
Special stains may be used at the discretion of the pathologist to further characterize lesions and changes identified during routine evaluation of individual animals. Any special stains will be documented in the individual animal data.
Statistics:
Body Weight Gains: Calculated against the body weight on Day 1 (pre-mating and lactation periods) or Day 0 (post-coitum period).
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions will be analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation will be reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics will be performed according to the matrix below when possible, but will exclude semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons will be made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Clinical signs:
no effects observed
Description (incidence and severity):
No test material-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gains of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight of treated animals was comparable to the control level over the treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test material-related changes were noted in hematology parameters.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test material.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related changes were noted in clinical chemistry parameters up to 300 mg/kg/day.
A lower cholesterol concentration was noted (0.71x of control, not statistically significant) for females treated at 1000 mg/kg/day. Individual values of 3/5 females were below the historical control range.
Any other changes in clinical chemistry parameters, regardless of reaching statistical significance, were considered to be unrelated to treatment with the test material as these occurred in the absence of a dose-related trend.
Endocrine findings:
no effects observed
Description (incidence and severity):
Serum levels of T4 in males were considered unaffected by treatment with the test material.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered unaffected by treatment with the test material.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
Motor activity was considered not to be affected by treatment with the test material. All groups showed a similar motor activity habituation profile with in general a decreasing trend in activity over the duration of the Test Period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test material-related alterations in organ weights.
The absolute weight of ovaries in females at 1000 mg/kg/day was statistically significant lower compared to the control group. This was considered incidental based on the lack of a dose response, the absence of a microscopic correlate and the fact that it was not significant when expressed relative to body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test material-related gross observations.
All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test material-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Details on results:
Wistar Han rats were treated with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received the vehicle, Corn Oil, alone.
No parental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
A lower concentration of cholesterol was noted for females treated at 1000 mg/kg/day. In absence of corroborative histopathological findings this was considered not adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
gross pathology
haematology
histopathology: neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)

CLINICAL SIGNS SUMMARY


MALES
















































 



 



PRE MATING



REPRO PERIOD



SIGN (MAX. GRADE)



WEEK:



1 . . . . . . . . . . . . .



1 . . . . . . . . . . . . .



(LOCATION)



DAY:



12345671234567



12345671234567



GROUP 1 (CONTROL)


No clinical signs noted



 



 



 



GROUP 2 (100 MG/KG/DAY)


No clinical signs noted



 



 



 



GROUP 3 (300 MG/KG/DAY)


No clinical signs noted



 



 



 



GROUP 4 (1000 MG/KG/DAY)


No clinical signs noted



 



 



 



G: Median value of the highest individual daily grades


%: Percent of affected animals (0=less than 5%, 1=between %% and 15%, …., A=more than 95%)


.: Observation performed, sign not present


 


CINICAL SIGNS SUMMARY


FEMALES
















































 



 



PRE MATING



REPRO PERIOD



SIGN (MAX. GRADE)



WEEK:



1 . . . . . . . . . . . . .



1 . . . . . . . . . . . . . . . . . . . .4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8



(LOCATION)



DAY:



12345671234567



12345671234567123456712345671234567123456712345671



GROUP 1 (CONTROL)


Skin / fur


            Alopecia (3)



 


 


G:


%:



 


 


. . . . . . . . . . . . . .


. . . . . . . . . . . . . .



 


 


. . . . . . . . . . . . . . . . . . . 111 . . . . . . . . . . . . . . . . . . . . .


. . . . . . . . . . . . . . . . . . . 111 . . . . . . . . . . . . . . . . . . . . .



GROUP 2 (100 MG/KG/DAY)


Skin / fur


            Piloerection (1)


 


            Alopecia (3)



 


 


G:


%:


G:


%:



 


 


. . . . . . . . . . . . . .


. . . . . . . . . . . . . .


. . . . . . . . . . . . . .


. . . . . . . . . . . . . .



 


 


. . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . .


. . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . .


. . . . . . . . . . . . . . . . . . . . . . . . . 111111111111111111 . .


. . . . . . . . . . . . . . . . . . . . . . . . . 111111111111111111 . .



GROUP 3 (300 MG/KG/DAY)


Skin / fur


            Alopecia (3)



 


 


G:


%:



 


 


. . . . . . . . . . . . . .


. . . . . . . . . . . . . .



 


 


. . . . . . . . . . . . . . . 111111111111111111111111 . . . . . . .


. . . . . . . . . . . . . . . 111111111111111111111111 . . . . . . .



GROUP 4 (1000 MG/KG/DAY)


Skin / fur


            Alopecia (3)



 


 


G:


%:



 


 


. . . . . . . . . . . . . .


. . . . . . . . . . . . . .



 


 


. . . . . . . . . . . . . . . . . . . . . 1111111111111111111 . . . . . . .


. . . . . . . . . . . . . . . . . . . . . 1111111111111111111 . . . . . . .



G: Median value of the highest individual daily grade


%: Percent of affected animals (0=less that 5%, 1=between 5% and 15%, …., A=more than 95%)


.: Observation performed, sign not present


 


BODY WEIGHTS (GRAM) SUMMARY


MALES




























































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



PRE MATING



DAY 1


WEEK 1



MEAN


ST.  DEV.


N



298


9.9


10



299


11.3


10



292


12.2


10



299


11.1


10



DAY 8


WEEK 2



MEAN


ST. DEV.


N



320


10.2


10



320


9.9


10



314


13.6


10



322


15.3


10



MATING PERIOD



DAY 1


WEEK 1



MEAN


ST. DEV.


N



342


11.4


10



338


10.9


10



331


15.3


10



340


16.0


10



DAY 8


WEEK 2



MEAN


ST. DEV.


N



353


12.0


10



346


10.2


10



340


17.2


10



350


8.8


10



DAY 15


WEEK 3



MEAN


ST. DEV.


N



368


13.2


10



360


14.5


10



354


17.9


10



363


19.7


10



FEMALES




















































































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



PRE MATING



DAY 1


WEEK 1



MEAN


ST.  DEV.


N



210


9.6


10



205


12.9


10



209


18.110



210


9.2


10



DAY 8


WEEK 2



MEAN


ST. DEV.


N



216


9.3


10



209


14.2


10



216


19.7


10



215


10.2


10



MATING PERIOD



DAY 1


WEEK 1



MEAN


ST. DEV.


N



221


10.2


10



215


16.9


10



220


19.9



219


10.3


10



DAY 8


WEEK 2



MEAN


ST. DEV.


N



 



 



230


12.9


3



243


--


1



DAY 15


WEEK 3



MEAN


ST. DEV.


N



 



 



263


--


1



 



DAY 22


WEEK 4



MEAN


ST. DEV.


N



 



 



246


--


1



 



DAY 29


WEEK 5



MEAN


ST. DEV.


N



 



 



242


--


1



 



DAY 36


WEEK 6



MEAN


ST. DEV.


N



 



 



242


--


1



 



*/**  Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


BODY WEIGHT GAIN (%) SUMMARY


MALES




























































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



PRE MATING



DAY 1


WEEK 1



MEAN


ST.  DEV.


N



0


0.0


10



0


0.0


10



0


0.0


10



0


0.0


10



DAY 8


WEEK 2



MEAN


ST. DEV.


N



8


1.2


10



7


1.6


10



7


1.3


10



7


1.4


10



MATING PERIOD



DAY 1


WEEK 1



MEAN


ST. DEV.


N



15


2.1


10



13


2.4


10



13


1.8


10



14


1.7


10



DAY 8


WEEK 2



MEAN


ST. DEV.


N



18


2.3


10



16


3.1


10



16


2.1


10



17


2.6


10



DAY 15


WEEK 3



MEAN


ST. DEV.


N



24


2.2


10



20


4.5


10



21


2.4


10



21


3.0


10



FEMALES




















































































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



PRE MATING



DAY 1


WEEK 1



MEAN


ST.  DEV.


N



0


0.0


10



0


0.0


10



0


0.0


10



0


0.0


10



DAY 8


WEEK 2



MEAN


ST. DEV.


N



3


2.3


10



2


1.3


10



4


1.6


10



2


1.8


10



MATING PERIOD



DAY 1


WEEK 1



MEAN


ST. DEV.


N



5


2.5


10



5


2.6


10



5


2.3


10



4


1.8


10



DAY 8


WEEK 2



MEAN


ST. DEV.


N



 



 



15


4.8


3



14


--


1



DAY 15


WEEK 3



MEAN


ST. DEV.


N



 



 



29


--


1



 



DAY 22


WEEK 4



MEAN


ST. DEV.


N



 



 



21


--


1



 



DAY 29


WEEK 5



MEAN


ST. DEV.


N



 



 



19


--


1



 



DAY 36


WEEK 6



MEAN


ST. DEV.


N



 



 



19


--


1



 



*/**  Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY


MALES




































































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



PRE MATING



DAYS 1-8


WEEKS 1-2



MEAN


ST. DEV.


N (CAGE)



23


0.1


2



22


0.0


2



23


0.0


2



23


1.2


2



DAYS 8-15


WEEKS 2-3



MEAN


ST. DEV.


N (CAGE)



22


0.4


2



22


0.1


2



22


0.3


2



22


0.8


2



MEAN OF MEANS OVER PRE MATING PERIOD



MEAN



23



22



22



23



MATING PERIOD



DAYS 1-8


WEEKS 1-2



MEAN


ST. DEV.


N (CAGE)



23


0.6


2



22


0.1


2



24


0.2


2



23


1.4


2



DAYS 8-15


WEEKS 2-3



MEAN


ST. DEV.


N (CAGE)



21


0.7


2



21


0.4


2



21


1.1


2



21


0.8


2



MEAN OF MEANS OVER MATING PERIOD



MEAN



22



21



23



22



FEMALES









































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



PRE MATING



DAYS 1-8


WEEKS 1-2



MEAN


ST. DEV.


N (CAGE)



15


0.9


2



14


0.4


2



16


0.4


2



16


0.9


2



DAYS 8-15


WEEKS 2-3



MEAN


ST. DEV.


N (CAGE)



16


1.2


2



15


0.6


2



16


0.6


2



16


0.7


2



MEAN OF MEANS OVER PRE MATING PERIOD



MEAN



15



15



16



16



 


FUNCTIONAL OBSERVATIONS SUMMARY


MALES









































































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



END OF TREATMENT



HEARING


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



PUPIL L


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



PUPIL R


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



STATIC R


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



STATIC R


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



GRIP FORE


GRAM



MEAN


ST. DEV.


N



1042


146


5



1179


240


5



1044


369


5



923


136


5



GRIP HIND


GRAM



MEAN


ST. DEV.


N



575


104


5



542


130


3



579


134


5



442


74


5



FEMALES









































































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



END OF TREATMENT



HEARING


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



PUPIL L


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



PUPIL R


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



STATIC R


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



STATIC R


SCORE 0/1



MEDIAN


N



0


5



0


5



0


5



0


5



GRIP FORE


GRAM



MEAN


ST. DEV.


N



1420


148


5



1354


106


5



1334


189


5



1327


255


5



GRIP HIND


GRAM



MEAN


ST. DEV.


N



857


185


5



882


292


5



721


61


5



720


158


5



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


+/++ Steel-test significant at 5% (+) or 1% (++) level


 


MOTOR ACTIVITY TEST SUMMARY


MALES




















Total Movements



GROUP 1


(CONTROL)



GROUP 2


(100 MG/KG/DAY)



GROUP 3


(300 MG/KG/DAY)



GROUP 4


(1000 MG/KG/DAY)



MEAN


N


ST. DEV



2896


5


970



2892


5


980



3352


5


672



2865


5


419



* indicates a p-value <0.05, 88 indicates a p-value <0.01


MEAN and ST. DEV values are calculated per group, from each animal’s total Total Movements over all intervals




















Ambulations



GROUP 1


(CONTROL)



GROUP 2


(100 MG/KG/DAY)



GROUP 3


(300 MG/KG/DAY)



GROUP 4


(1000 MG/KG/DAY)



MEAN


N


ST. DEV



622


5


340



592


5


209



695


5


228



610


5


182



* indicates a p-value <0.05, 88 indicates a p-value <0.01


MEAN and ST. DEV values are calculated per group, from each animal’s total Ambulations over all intervals


 


MOTOR ACTIVITY TEST SUMMARY


FEMALES




















Total Movements



GROUP 1


(CONTROL)



GROUP 2


(100 MG/KG/DAY)



GROUP 3


(300 MG/KG/DAY)



GROUP 4


(1000 MG/KG/DAY)



MEAN


N


ST. DEV



3686


5


1400



3741


5


646



2833


5


867



4026


5


1483



* indicates a p-value <0.05, 88 indicates a p-value <0.01


MEAN and ST. DEV values are calculated per group, from each animal’s total Total Movements over all intervals




















Ambulations



GROUP 1


(CONTROL)



GROUP 2


(100 MG/KG/DAY)



GROUP 3


(300 MG/KG/DAY)



GROUP 4


(1000 MG/KG/DAY)



MEAN


N


ST. DEV



817


5


337



745


5


220



586


5


133



890


5


353



* indicates a p-value <0.05, 88 indicates a p-value <0.01


MEAN and ST. DEV values are calculated per group, from each animal’s total Ambulations over all intervals


 


MACROSCOPIC FINDINGS SUMMARY


MALES




















 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



END OF TREATMENT


Animals examined


Animals without findings


 


Animals affected


 


Stomach


            Focus/foci


Mesenteric lymph node


            Focus/foci



 


10


10


 


0


 


 


0


 


0



 


10


10


 


0


 


 


0


 


0



 


10


9


 


1


 


 


1


 


0



 


10


8


 


2


 


 


0


 


2



FEMALES




















 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



END OF TREATMENT


Animals examined


Animals without findings


 


Animals affected


 


Liver


            Diaphragmatic hernia


            Reduced in size


            Discolouration


Clitoral glands


            Discolouration


Thymus


            Focus/Foci


Mesenteric lymph node


            Focus/foci



 


10


10


 


0


 


 


0


0


0


 


0


 


0


 


0



 


10


9


 


1


 


 


0


0


0


 


0


 


1


 


0



 


10


7


 


3


 


 


1


1


1


 


1


 


0


 


0



 


10


9


 


1


 


 


0


0


0


 


0


 


0


 


1



# / ## Fisher’s Exact test significant at 5% (#) or 1 % (##) level


 


ORGAN WEIGHTS (GRAM) SUMMARY


MALES

























































































































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



END OF TREATMENT



BODY W.


(GRAM)



MEAN


ST. DEV


N



348


13


10



338


12


10



332


18


10



343


20


10



BRAIN


(GRAM)



MEAN


ST. DEV


N



2.02


0.05


5



2.07


0.07


5



2.00


0.03


5



1.98


0.07


5



HEART


(GRAM)



MEAN


ST. DEV


N



0.937


0.053


5



0.992


0.096


5



0.969


0.064


5



0.998


0.090


5



LIVER


(GRAM)



MEAN


ST. DEV


N



8.72


1.08


5



8.49


0.59


5



8.40


0.56


5



8.82


0.78


5



THYROIDS


(GRAM)



MEAN


ST. DEV


N



0.0170


0.0036


10



0.0155


0.0038


10



0.0149


0.0026


10



0.0160


0.0017


10



THYMUS


(GRAM)



MEAN


ST. DEV


N



0.341


0.067


5



0.362


0.101


5



0.367


0.067


5



0.328


0.041


5



KIDNEYS


(GRAM)



MEAN


ST. DEV


N



2.16


0.27


5



2.27


0.12


5



2.28


0.10


5



2.12


0.20


5



ADRENALS


(GRAM)



MEAN


ST. DEV


N



0.055


0.011


5



0.062


0.007


5



0.059


0.007


5



0.059


0.015


5



SPLEEN


(GRAM)



MEAN


ST. DEV


N



0.584


0.093


5



0.546


0.034


5



0.560


0.080


5



0.573


0.097


5



TESTES


(GRAM)



MEAN


ST. DEV


N



3.41


0.19


10



3.51


0.23


10



3.39


0.19


10



3.43


0.17


10



PROSTATE GLAND


(GRAM)



MEAN


ST. DEV


N



0.755


0.132


10



0.739


0.131


10



0.750


0.095


10



0.703


0.120


10



EPIDIDYMIDES


(GRAM)



MEAN


ST. DEV


N



1.050


0.069


10



1.042


0.066


10



1.019


0.052


10



1.053


0.052


10



SEMINAL VESICLES


(GRAM)



MEAN


ST. DEV


N



1.218


0.130


10



1.207


0.184


10



1.181


0.310


10



1.192


0.146


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


ORGAN/BODY WEIGHT RATIOS (%) SUMMARY


MALES

























































































































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



END OF TREATMENT



BODY W.


(GRAM)



MEAN


ST. DEV


N



348


13


10



338


12


10



332


18


10



343


20


10



BRAIN


(%)



MEAN


ST. DEV


N



0.59


0.03


5



0.61


0.03


5



0.59


0.02


5



0.57


0.05


5



HEART


(%)



MEAN


ST. DEV


N



0.271


0.014


5



0.290


0.022


5



0.287


0.015


5



0.283


0.010


5



LIVER


(%)



MEAN


ST. DEV


N



2.52


0.22


5



2.48


0.14


5



2.49


0.12


5



2.50


0.06


5



THYROIDS


(%)



MEAN


ST. DEV


N



0.0049


0.0009


10



0.0046


0.0011


10



0.0045


0.0009


10



0.0047


0.0006


10



THYMUS


(%)



MEAN


ST. DEV


N



0.098


0.015


5



0.106


0.028


5



0.109


0.024


5



0.093


0.009


5



KIDNEYS


(%)



MEAN


ST. DEV


N



0.62


0.06


5



0.66


0.03


5



0.68


0.06


5



0.60


0.03


5



ADRENALS


(%)



MEAN


ST. DEV


N



0.016


0.003


5



0.018


0.001


5



0.017


0.002


5



0.017


0.005


5



SPLEEN


(%)



MEAN


ST. DEV


N



0.169


0.023


5



0.160


0.012


5



0.165


0.020


5



0.163


0.023


5



TESTES


(%)



MEAN


ST. DEV


N



0.98


0.08


10



1.04


0.08


10



1.02


0.08


10



1.00


0.07


10



PROSTATE GLAND


(%)



MEAN


ST. DEV


N



0.217


0.039


10



0.219


0.042


10



0.225


0.019


10



0.205


0.032


10



EPIDIDYMIDES


(%)



MEAN


ST. DEV


N



0.303


0.026


10



0.308


0.024


10



0.307


0.020


10



0.308


0.027


10



SEMINAL VESICLES


(%)



MEAN


ST. DEV


N



0.351


0.039


10



0.358


0.057


10



0.356


0.095


10



0.349


0.048


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


ORGAN WEIGHTS (GRAM) SUMMARY


FEMALES









































































































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



END OF TREATMENT



BODY W.


(GRAM)



MEAN


ST. DEV


N



289


20


10



273


32


10



276


33


9



282


21


8



BRAIN


(GRAM)



MEAN


ST. DEV


N



1.86


0.04


5



1.89


0.06


5



1.87


0.06


5



1.87


0.03


5



HEART


(GRAM)



MEAN


ST. DEV


N



0.865


0.057


5



0.827


0.062


5



0.833


0.125


5



0.806


0.040


5



LIVER


(GRAM)



MEAN


ST. DEV


N



12.37


1.51


5



11.79


0.96


5



12.36


1.61


5



11.93


1.34


5



THYROIDS


(GRAM)



MEAN


ST. DEV


N



0.0140


0.0024


10



0.0144


0.0024


10



0.0145


0.0026


10



0.0140


0.0020


10



THYMUS


(GRAM)



MEAN


ST. DEV


N



0.196


0.0304


5



0.186


0.035


5



0.198


0.015


5



0.185


0.027


5



KIDNEYS


(GRAM)



MEAN


ST. DEV


N



1.87


0.15


5



1.91


0.09


5



1.92


0.27


5



1.86


0.15


5



ADRENALS


(GRAM)



MEAN


ST. DEV


N



0.082


0.014


5



0.072


0.007


5



0.074


0.015


5



0.074


0.010


5



SPLEEN


(GRAM)



MEAN


ST. DEV


N



0.507


0.074


5



0.500


0.037


5



0.499


0.015


5



0.525


0.050


5



OVARIES


(GRAM)



MEAN


ST. DEV


N



0.126


0.022


5



0.117


0.014


5



0.123


0.020


5



0.097*


0.011


5



UTERUS


(GRAM)



MEAN


ST. DEV


N



0.343


0.027


5



0.322


0.057


5



0.356


0.022


5



0.354


0.040


5



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


ORGAN/BODY WEIGHT RATIOS (%) SUMMARY


FEMALES









































































































 



 



GROUP 1


CONTROL



GROUP 2


100


MG/KG/DAY



GROUP 3


300


MG/KG/DAY



GROUP 4


1000


MG/KG/DAY



END OF TREATMENT



BODY W.


(GRAM)



MEAN


ST. DEV


N



289


20


10



273


32


10



276


33


9



282


21


8



BRAIN


(%)



MEAN


ST. DEV


N



0.66


0.05


5



0.67


0.06


5



0.67


0.07


5



0.69


0.05


5



HEART


(%)



MEAN


ST. DEV


N



0.306


0.021


5



0.291


0.016


5



0.296


0.015


5



0.295


0.015


5



LIVER


(%)



MEAN


ST. DEV


N



4.36


0.39


5



4.14


0.07


5



4.39


0.15


5



4.36


0.35


5



THYROIDS


(%)



MEAN


ST. DEV


N



0.0049


0.0011


10



0.0054


0.0012


10



0.0052


0.0012


9



0.0050


0.0010


8



THYMUS


(%)



MEAN


ST. DEV


N



0.070


0.015


5



0.066


0.015


5



0.071


0.006


5



0.068


0.010


5



KIDNEYS


(%)



MEAN


ST. DEV


N



0.66


0.05


5



0.67


0.03


5



0.68


0.03


5



0.68


0.04


5



ADRENALS


(%)



MEAN


ST. DEV


N



0.029


0.005


5



0.025


0.001


5



0.026


0.003


5



0.027


0.004


5



SPLEEN


(%)



MEAN


ST. DEV


N



0.179


0.025


5



0.176


0.015


5



0.179


0.034


5



0.192


0.008


5



OVARIES


(%)



MEAN


ST. DEV


N



0.045


0.009


5



0.041


0.005


5



0.046


0.012


6



0.036


0.005


5



UTERUS


(%)



MEAN


ST. DEV


N



0.122


0.014


5



0.115


0.028


5



0.129


0.024


5



0.130


0.018


5



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 

Conclusions:
In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid were established:
Parental NOAEL: at least 1000 mg/kg/day
Executive summary:

The objectives of this study were to determine the potential toxic effects of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.


In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.


 


The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the Dose Range Finder.


 


The study design was as follows:


Experimental design




















































Group No.



Test item Identification



Dose Level (mg/kg/day)



Dose Volume (mL/kg)



Dose Concentration (mg/mL)



Number of Animals



Males



Females



1



-



0 (vehicle)



4



0



10



10



2



Decanoic acid, mixed ester with dipentaerythritol, octanoic acid and valeric acid



100



4



25



10



10



3



300



4



75



10



10



4



1000



4



250



10



10



 


Chemical analyses of formulations were conducted once during the study and confirmed that formulations were prepared accurately and homogenously.


 


The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and food consumption, functional observations, clinical pathology (hematology, coagulation, clinical chemistry and thyroid hormone analysis (T4 in F0-males), macroscopic examination, organ weights and microscopic examination.


 


At 1000 mg/kg/day, non-adverse lower cholesterol concentration were noted in females. No test material-related changes were noted in any of the remaining parameters investigated in this study (i.e., mortality, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, hematology, clotting parameters, thyroid hormone analysis (T4 thyroid hormone levels in F0-males), macroscopic examination, organ weights, and microscopic examination).


 


In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) for Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid were established:


Parental NOAEL: at least 1000 mg/kg/day

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Remarks:
Study on analogue; see RAAF attached.
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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
see below
Qualifier:
according to guideline
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
Species:
rat
Strain:
other: CD® [Crl:CD®(SD)]
Sex:
male/female
Details on test animals or test system 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.
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.
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.
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).
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.
Key result
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.
Key result
Critical effects observed:
not specified

   

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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
7
Species:
rat
Quality of whole database:
1

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

This endpoint is read across to the structural analogue.  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.

Justification for classification or non-classification

Based on the above mentioned result, classification according to the CLP Regulation (EC) 1272/2008.