Reaction product of Fatty acids, C18 alkyl with amines, polyethylenepoly-tetraethylenepentamine fraction

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment Start Date : 30 April 2020 and Study completion : 26 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Guideline adopted on June 25, 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

High moecular weight adducts of Fatty acids, C16-18 sat. C18 unsat., linear, dimers, and trimers, with Amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

High moecular weight adducts of Fatty acids, C16-18 sat. C18 unsat., linear, dimers, and trimers, with Amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 2

Reference substance name:

Higher molecular weight adducts of C18 Fatty acids linear (unsat & sat) with amines,polyethylenepoly-, tetraethylenepentamine fraction

IUPAC Name:

Higher molecular weight adducts of C18 Fatty acids linear (unsat & sat) with amines,polyethylenepoly-, tetraethylenepentamine fraction

Constituent 3

Reference substance name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

EC Number:

292-587-7

EC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Cas Number:

90640-66-7

IUPAC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Constituent 4

Reference substance name:

lower molecular weight adducts of C18 Fatty acids linear (unsat & sat) amines,polyethylenepoly-, tetraethylenepentamine fraction

Molecular formula:

for example : C26H53N5O, C26H55N5O...

IUPAC Name:

lower molecular weight adducts of C18 Fatty acids linear (unsat & sat) amines,polyethylenepoly-, tetraethylenepentamine fraction

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report):TOFA DimerFA TEPA PAA
- Physical state:Yellow-red, transparent, viscous liquid
- Analytical purity: 9% free amine
- Storage condition of test material:Room temperature (15°C to 30°C)
-Sponsor batch: BB000649V1

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source : Hylasco Biotechnology (India) Pvt. Ltd. 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
- Age at study initiation: 5 weeks
- Females (if applicable) nulliparous and non-pregnant: YES
- Weight at study initiation: Males : 173.77 to 223.03g and Females : 138.70 to 183.41g
- Fasting period before study: not specified
- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfonecages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill havingfacilities for pelleted food and drinking water in polycarbonate bottles with
stainless steel sipper tubes. The last animal in recovery group of each sex warhoused individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area (SC-22).
- Diet (e.g. ad libitum): ad libitum. Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, will be provided
- Water (e.g. ad libitum): Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, will be provided ad libitum
- Acclimation period: 5 days before the start of the testing. During acclimatization period, rats were observed once daily for any abnormalities. Nulliparous and non-pregnant females were used in the study.

DETAILS OF FOOD AND WATER QUALITY: The feed and water provided to the rats was tested for contaminants.
Based on the latest analytical certificate/s available, there were no known contaminants in the food, water and bedding that are expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 21 to 24°C
- Humidity (%): between 49 to 68 %
- Air changes (per hr): 12.5-12.8 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle


IDENTIFICATION : Temporary: All rats were identified using cage card and crystal violet solution during acclimatization. Permanent: All rats were identified by rat accession number, cage card and tail tattooing (tail tattooing was done six days after grouping). Turmeric yellow
body marking was done on the day of grouping and animals were not re-marked thereafter.


IN-LIFE DATES: From: To:
*06 May 2020 Dose formulation preparation and administration by oral gavage – Day 1. Dose formulation analysis: Sample preparation and processing – Day1.

*03 June 2020 Body weights, feed output and input measurement and dose formulation administration by oral gavage – Day 29

*01 July 2020 Detailed clinical examination – Day 57, Week 9

*14 July 2020 Dose formulation analysis: Processing of dose formulation samples

*04 August 2020 Necropsy

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 90 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once orally for 90 consecutive days.
The vehicle or the dose formulations were not administered to recovery groups for 28 days following the 90-day treatment period.
The dose formulation and the vehicle were administered at an equivolume of 5 mL/kg/day. The dose volume was calculated for individual animals on the first day of treatment and was adjusted according to the most recent body weights recorded during the treatment period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd (Day 35) and 3rd (Day 69) month of the treatment period and analysed in-house.
For each set, duplicate samples from top, middle and bottom layers were drawn from each preparation and in case of control, duplicate samples were drawn from middle layer.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G18456. One set of samples were analyzed for concentration analysis and other set (second set) of samples were stored at ambient condition at Analytical R&D department for reanalysis purpose as a backup and the second set of samples were discarded, as the analysis results of first set of samples are within the limits.

Formulations were considered acceptable as overall mean results of all layers and mean of each layer were within ± 20 % of the claimed concentration and the overall relative standard deviation (RSD) was less than 15 %.

The results indicated the percent agreement of the analyzed concentrations of all the layers and mean of each layer were in the range, 80% to 120% of the claimed concentrations. The relative standard deviation (RSD) was less than 15.0%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle.corn oil Vehicle control (G1) / vehicle control recovery (G1R)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose (G2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid dose (G3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose (G4) / high dose recovery (G4R)

No. of animals per sex per dose:

Each main group in the experiment was comprised of 10 male and 10 female rats and recovery groups comprised of 5 male and 5 female rats.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose level and Dose justification
The dose levels of 100 (G2), 300 (G3) and 1000 (G4/G4R) mg/kg/day were selected for this study based on the results of 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No.N4575) and in consultation with the Sponsor.
In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and recovery were handled in a manner similar to the treatment groups except for test item administration.
In 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats, the dose levels of 100, 300 and 1000 mg/kg bwt/day were used. In brief, the relevant results are as follows;
Clinical Signs and Mortality: There were no clinical signs or mortalities observed at all the tested doses.

*Body weights and Food Consumption: The mean body weight, body weight gains and food consumption were comparable to vehicle control group.
*Hematology: Increase in WBC count with increased neutrophil count at 1000 mg/kg/day in both genders and at 300 mg/kg/day in females.
*Coagulation: The treatment did not induce any test item-related changes in coagulation parameters at any of the doses tested in both sexes.
*Clinical Chemistry: Elevated ALT and AST enzymes levels at 1000 mg/kg/day dose in both males and females and ALT at 300 mg/kg/day dose in males. In the absence of organ weights and their ratios, the changes observed at 300 mg/kg/day were considered treatment related non-adverse changes.
*Terminal Fasting Body Weights and Organ Weights: The terminal body weights were not affected by the test item treatment. Organ weights showed lower thymus weight at 1000 mg/kg/day in males and females.
*Gross pathology: Grossly, non-glandular thickening of stomach was noted in females at 1000 mg/kg/day.




- Rationale for animal assignment (if not random):
Rats were randomly distributed to different groups by the body weight stratification method using ProvantisTM Software (Version 10.1.0.1, Instem LSS, Staffordshire ST15OSD, United Kingdom). Rats with extreme body weights were excluded from the study. Grouping was done during
acclimatization period.

Positive control:

not appicable

Examinations

Observations and examinations performed and frequency:

Morbidity and Mortality
All rats were observed for morbidity and mortalities twice daily i.e., once in themorning and once in the afternoon except during holidays wherein the observation was done once daily as there were no clinical signs observed.

Clinical Signs
Each rat was observed for checking general clinical signs twice once daily during treatment period and once daily during the recovery period. On the days of scheduled detailed clinical examination, clinical signs were included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the
treatment and observations for general clinical signs was done after dosing the animals.


Detailed Clinical Examination
Detailed clinical examination was done prior to the test item administration onDay 1 and at weekly intervals thereafter (± 1 days) during treatment period. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory
pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards).


Ophthalmological Examination
Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups (Day 90) and at the end of recovery period (Day 115) for recovery groups. Before examination, mydriasis was induced using a 1 % solution of Tropicamide.

Functional Observation Battery Tests (FOB)
The following neurological examination was performed during the 12th week (Day 85) of treatment period for main groups and towards the end of recovery period (Day 116) for recovery groups.


Home Cage Observations
Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
8.5.2 Observations during Removal of Animal from Home Cage and Handling The objective of this phase of neurological examination was to observe the subject’s response to handling and to other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response
The observations were recorded using scores/ranks.


Open Field Observation
Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, rat was evaluated as it moves about freely/unperturbed and the following observations were made and observations were recorded using score/ranks:
- gait
- posture
- tremors
- mobility score
- arousal level
- clonic or tonic movements
- stereotypic behaviour
- bizarre behaviour
- urination
- defecation
- rearing
- abnormal vocalizations


Functional Tests
Functional testing included motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.


Motor Activity
The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements)
in seconds, the ambulatory time (large ambulatory movement) in seconds, distance travelled in centimeter and resting time in seconds were monitored. The Opto-Varimex 5 data was analyzed in 10 minutes intervals and the same was reported.


Sensory Reactivity Measurements
After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were
recorded using scores/ranks.
- approach response
- touch response
- click response
- tail-pinch response
- pupil response
- aerial righting reflex

Landing Hindlimbs Footsplay
The landing hind limbs foot splay was performed by dropping the rat onto a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat was gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP
format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values were is presented in the report along with the individual footsplay values.


Grip Performance
Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Averages of three trials for both forelimb and hindlimbs were calculated and presented in the report along with the individual grip strength values.


Physiological Observations
Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured.


Body Weight
Individual body weights (g) were recorded prior to test item administration on Day 1 and weekly thereafter (± 2 day) for all groups of rats during treatment and recovery period. Fasting body weight was recorded prior to sacrifice for all animals.


Food Consumption
The food consumption was measured at weekly intervals (± 2 days) during treatment and recovery period. The cage wise average food consumption (g/rat/day) was calculated and presented in the report.


Oestrous Cycle Evaluation
Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.

Sacrifice and pathology:

Anatomic Pathology
Necropsy
All the rats from main group were sacrificed at end of the treatment period (Day 91) and end of recovery period (Day 119). All animals were fasted overnight (water allowed), weighed before necropsy and euthanized under isofluranev anesthesia. Detailed macroscopic examination was performed on all animals including external features of the carcass, external body orifices, abdominal, thoracic, and cranial cavities, organs and tissues and abnormalities were recorded.

Tissue Collection, Weighing and Preservation
On completion of the gross pathology examination, the tissues and organs noted below were collected and weighed from all animals. Organ weight ratios as percentage of body weight (using the terminal body weight obtained at necropsy) and brain weight were determined and presented in the report. The paired organs were weighed together and combined weight was presented. Tissues were preserved in 10% Neutral Buffered Formalin (NBF) except for the testes and eyes:

Histopathology
Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose group animals (G4) including all gross lesions. The testes sections were also examined for the qualitative assessment of stages of spermatogenesis. Further, following organs suspected of showing test item-related histopathological changes in high dose (G4) group were examined in the respective lower dose groups and recovery groups.

Males- Liver, stomach, mesenteric lymph nodes, spleen, thymus, adrenals, testes, epididymides, seminal vesicles, coagulating gland, prostate, sternum with marrow, femur bone and bone marrow smears.

Females- Liver, stomach, mesenteric lymph nodes, spleen, thymus, adrenals, sternum with marrow, femur bone and bone marrow smears.

The tissues were processed for routine paraffin embedding and 4-5 micron sections were stained with Haematoxylin and Eosin stain. Unused tissues will be archived. Additional section of testes (4 micron) was stained with Periodic Acid Schiff reagent and hematoxylin to have the detailed examination on stages of spermatogenesis

Other examinations:

Blood Collection
At the end of the treatment and recovery periods, all rats were fasted overnight (water allowed) and approximately 4.0 mL of blood was collected (as indicated in the following Table) under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture.

Haematology
1 Red Blood Corpuscles
2 Haemoglobin
3 Haematocrit
4 Mean Corpuscular
5 Mean Corpuscular Haemoglobin
6 Mean Corpuscular Haemoglobin
7 Reticulocytes count
8 White Blood Corpuscles
9 Differential leukocyte count
10 Platelets

Additionally, blood smears were prepared from the haematology (K2EDTA tube) samples and stained with Giemsa stain. Examination of smears was not done by conventional microscopy as hematology findings did not warrant examination. All blood smear slides will be discarded at the time of final report preparation.

Coagulation
Blood samples collected for coagulation analysis were centrifuged at 2500 times gravity (xg) for 10 minutes at 15ºC for separation of plasma. The plasma samples were analyzed for the following parameters using Start Max coagulation analyzer (Diagnostica stago, 92600 Asnieres, France):

1 Prothrombin Time
2 Activated Partial Thromboplastin Time

Clinical Chemistry
Plasma was separated after centrifugation of the whole blood samples at 4ºC, 5000 rpm for 5 minutes and analyzed using Dimension RxL MaX Clinical Chemistry System (Dade Behring Inc. Newark, DE 19714, USA) for the following parameters:
Alanine Aminotransferase ALT U/L
2 Alkaline Phosphatase Alp U/L
3 Albumin ALB g/L
4 Albumin/Globulin ratio (calculated value) A/G -
5 Aspartate Aminotransferase AST U/L
6 Blood Urea Nitrogen BUN mmol/L
7 Chloride Cl mEq/L
8 Creatinine Creat µmol/L
9 Calcium Ca mmol/L
10 Glucose Glu mmol/L
11 Globulin (calculated value) GLOB g/L
12 HDL Cholesterol HDL.Chol mmol/L
13 Inorganic Phosphorous Pi mmol/L
14 LDL Cholesterol2 LDL.Chol mmol/L
15 Potassium K mEq/L
16 Sodium Na mEq/L
17 Total Cholesterol T. Chol mmol/L
18 Total Plasma Protein T. Pro g/L
19 Triglycerides Trig mmol/L
20 Total Bilirubin T. Bil µmol/L
21 Direct Bilirubin1 D.Bil µmol/L
22 Indirect Bilirubin (calculated)

Hormone Analysis
At the end of the treatment and recovery periods, blood was collected from all rats along with blood collection for clinical pathology investigation for the determination of total T3, T4 and TSH hormones.

Blood samples were collected in plain labelled tubes and kept on bench top for 30 min before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4° C. The serum samples were placed in labelled plastic tubes and stored at ~ -70 °C until they were analyzed.
The left-over samples from hormone analysis were discarded after data review.

Thyroid Profile Hormones:
The following thyroid hormones were estimated by Enzyme Linked Immuno Sorbent Assay (ELISA) method using BIO-RAD microplate washer and BIORAD model 680 readers.

1 Rodent Thyroid Stimulating Hormone TSH ng/mL
2 Rodent Thyroxine T4 ng/mL
3 Rodent Triiodothyronine


Urinalysis
Urine was collected from all rats at the end of the treatment period (Day 91) and recovery period (Day 119) in urine collection tube. Each rat was placed in a specially fabricated cage (water allowed) for about 3-4 hours and the collected urine was analyzed for the following parameters.
Urine was analyzed for:

1 Specific gravity1
2 Nitrite2
3 pH2
4 Proteins2
5 Glucose2
6 Ketone bodies2
7 Urobilinogen2
8 Bilirubin2
9 Appearance (colour and clarity)3

Statistics:

Data captured using Provantis™ for the parameters body weight and organ weights; laboratory Investigations – Haematology, Coagulation and Clinical Chemistry were analyzed using built-in statistical tests.

Derived data like net body weight change, food consumption and organ weight ratios were also analyzed using above mentioned methods.

The statistical analysis of neurological observations (neuromuscular observation/body temperature/body weights) and the hormones, viz. TSH, T4, T3 data were carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. The data were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before performing ANOVA. Comparison of means between treatment and control groups was done using Dunnett’s test when the overall treatment, ‘F’ test found significant.

The data pertaining to males and female rats was evaluated separately. All analyses and comparisons were evaluated at the 5% (p<0.05) level.
Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated by the following symbols throughout the report:
*: Significantly higher/lower than the respective control group

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed during the treatment at 100 mg/kg bwt/day dose group.
Transient clinical sign of slight salivation was observed soon after the dose administration in all animals at 1000 mg/kg bwt/day starting from treatment Day 5 and at 300 mg/kg bwt/day starting from treatment Day 45. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. During the recovery period no clinical signs observed.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed during the treatment at 100 mg/kg bwt/day dose group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
At 1000 mg/kg/day, the mean body weights were significantly lower on Days 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90, compared to vehicle control group.
The absolute body weight gain was also significantly lower during Days 1-8, 22-29, 78-85 and 1-90 of the treatment period, compared to vehicle control group.

The absolute weight gain was significantly higher during Days 104-111 at 1000 mg/kg/day recovery group and toxicologically not significant.

At 100 and 300 mg/kg/day, the treatment did not affect the mean body weights and net body weight gains, compared to vehicle control group. However, incidence of significantly lower absolute weight gain during Days 36-43 at 300 mg/kg/day were considered toxicologically not significant.

Females: The body weights and body weight gains were unaffected by the treatment at all the doses tested, both during treatment and recovery periods.
However, the absolute weight gain was significantly higher during Days 43-50 at 1000 mg/kg/day recovery group and significantly lower during Days 22-29 at 1000 mg/kg/day main groups and significantly lower during Days 8-15 at 1000 mg/kg/day recovery group. The incidence of significantly lower absolute weight gain during Days 64-71at 300 mg/kg/day was observed. These significant differences were not considered toxicologically relevant as the mean body weights and total body weight gains were not altered by the treatment.

Thus, the treatment decreased the body weights in males at 1000 mg/kg/day, however, the body weights were unaffected at 100 and 300 mg/kg/day in males and at all the doses tested in females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males: The food consumption was significantly lower throughout the treatment (expect during Days 15-22 and 36-43) at 1000 mg/kg/day. At 100 and 300 mg/kg/day, the food consumption was significantly lower during Days 1-8, 15-22, 22-29, 36-43, 50-57, 57-64, 64-71, 78-85, and 85-90. Significantly lower food consumption were observed during Days 29-36 and 71-78 at 300 mg/kg/day and during Days 43-50 at 100 mg/kg/day, compared to vehicle control group.
At 1000 mg/kg/day dose recovery group, the food consumption was significantly lower during Days 1-8, 8-15, 15-22, 36-43, 78-85 and 85-90, compared to concurrent vehicle control recovery group.

Females: The food consumption was significantly lower during Days 50-57, 64-71, 78-85 and 85-90 at 1000 mg/kg/day. At 100 and 300 mg/kg/day, the food consumption was significantly lower during Days 36-43, 50-57, 64-71, 78-85 and 85-90. Significantly lower food consumption were observed during Days 29-36 at 300 mg/kg/day and during Days 15-22 at 100 mg/kg/day, compared to vehicle control group.
At 1000 mg/kg/day dose recovery group, the food consumption was significantly lower during Days 1-8, 8-15, 85-90 and higher during Days 104- 111 and 111-118.

These significant differences were not considered toxicologically relevant as the mean body weights were not altered by the treatment and/or dose dependency was not observed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examination carried out with an ophthalmoscope prior to start of treatment, at the end of the treatment and recovery period did not reveal any abnormalities in the eyes of the experimental rats

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item related increase in total leukocyte count, neutrophils and monocytes in both males and females at 1000 mg/kg/day correlated with increased extramedullary hematopoiesis in spleen and adrenals and increased cellularity in bone marrow (sternum and femur). Leukocytes parameter changes were reversible at the end of the recovery period. Test item related decrease in total protein/albumin/globulin, triglycerides, cholesterol and AHDL were noted in both males and females at 1000 mg/kg/day. All these changes were completely reversible except for the reduced protein profile in females at the end of the recovery period. All the changes in leukocytes parameters and protein profile were secondary to the local inflammatory condition (mucosal necrosis) noted in the non-glandular stomach and these systemic or local changes were considered non-adverse. Increased ALT levels were noticed in both sex at ≥ 300 mg/kg/day without any correlating microscopic lesions in the liver, which was considered as a non-adverse effect of the test item.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The decreased terminal fasting body weight was noticed in males (18%) at 1000 mg/kg/day and was reversible. At 1000 mg/kg/day, increased adrenal weights (microscopically, associated with increased extramedullary hematopoiesis); decreased thymus weights (grossly, small and microscopically, associated with decreased cellularity-cortex/medulla in few males and females) and decreased spleen (microscopically, decreased cellularity-marginal zone of white pulp in two males) and prostate weights (microscopically, atrophy with decreased secretion in a male) were noticed. Reductions in weight of spleen, thymus, heart and prostate and increased brain weights in males at 1000 mg/kg/day were considered secondary effects due to either stress or decreased feed intake and/or reduced terminal body weight. The reduced thymus weight was also noted in males at 300 mg/kg/day without any histological correlation. These organ weight changes were not considered as adverse.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Grossly, thickening of non-glandular mucosa (stomach) at 1000 mg/kg/day males and females was histologically accompanied by epithelial hyperplasia/ mucosal necrosis. The range of microscopic changes were suggestive of forestomach gastritis specifically observed in rodents and was attributed to possible irritant potential of the test item.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No treatment related neurological abnormalities/dysfunctions were observed at all doses tested.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, degeneration of seminiferous tubules of testes (grossly- bilateral flabby testes in a male ) and atrophy of accessory
sex glands- prostate, seminal vesicles and coagulating gland (grosslyprostate-small in a male) in males at 1000 mg/kg/day were noted. Epididymal cell debris were considered consequent to degenerative changes noted in testes. These changes were attributed to the significant body weight reduction observed in these animals and considered test item related secondary effects. Microscopically, increased macrophages in mesenteric lymph nodes (in cortex and paracortex region); increased extramedullary hematopoiesis in spleen (in red pulp)/adrenals (inner cortical region-zona reticularis) and increased cellularity in bone marrow of sternum/femur/marrow smear in both sex were noted at ≥ 100 mg/kg/day.
The increased macrophages/extramedullary hematopoiesis/cellularity were also observed in recovery rats at the 28 day recovery period.

All the above changes in pathology parameters were considered either as local response (stomach lesions) or secondary changes associated with the decreased body weight /stomach lesions and thus not considered as adverse.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Hormone Profile: Thyroid hormone profile (TSH, T4 and T3) was not affected in both sexes across the treated groups when compared to the concurrent vehicle control group.

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Days Relative to start date	Male Body Weight (G)	G1 0 mg/kg/d	G2 100 mg/kg/d	G3 300 mg/kg/d	G4 1000 mg/kg/d
1[a]	Mean SD N	196.62 16.22 10	196.24 15.61 10	194.92 16.72 10	194.00 14.33 10
8[a]	Mean SD N	258.06 18.97 10	253.67 16.45 10	255.18 19.39 10	249.05 14.77 10
15[a1]	Mean SD N	314.23 26.23 10	312.86 15.39 10	310.98 26.10 10	299.80 18.20 10
22[a1]	Mean SD N	350.14 26.23 10	352.24 19.78 10	344.44 27.19 10	344.56 19.39 10
29[a]	Mean SD N	383.50 31.02 10	382.99 22.89 10	373.96 34.91 10	345.40* 28.22 10
36[a]	Mean SD N	409.20 36.62 10	413.08 24.55 10	402.88 37.13 10	357.89* 32.37 10
43[a]	Mean SD N	441.89 46.28 10	436.41 28.50 10	421.34 40.76 10	379.90* 37.94 10
50[a]	Mean SD N	461.96 48.90 10	462.37 27.48 10	445.82 44.08 10	409.57* 40.30 10
57[a]	Mean SD N	484.04 49.97 10	481.33 27.48 10	459.62 43.29 10	432.86* 34.57 10
64[a]	Mean SD N	504.73 54.19 10	499.50 30.22 10	476.86 38.82 10	440.31* 37.48 10
71[a]	Mean SD N	524.48 58.82 10	516.38 32.34 10	490.90 40.45 10	459.61* 43.82 10
78[a]	Mean SD N	538.33 58.73 10	534.73 36.01 10	505.98 42.21 10	474.82* 47.82 10
85[a]	Mean SD N	552.90 61.17 10	547.30 35.81 10	515.58 43.34 10	477.11* 54.09 10
90[a]	Mean SD N	556.03 59.00 10	554.44 39.61 10	519.64 50.28 10	466.93* 71.41 10

[a] anova & dunnet *=p<0.05

[a1] anova & dunnett (rank)

Days relative to start date	Female Body weight (G)	G1  0 mg/kg/d	G2  100 mg/kg/d	G3 300 mg/kg/d	G4 1000 mg/kg/d
1[a]	Mean  SD N	155.93 9.48 10	156.59 11.92 10	157.40 12.71 10	155.10 11.51 10
8[a]	Mean SD N	187.29 11.85 10	186.06 11.33 10	184.36 16.47 10	182.59 8.42 10
15[a]	Mean SD N	214.85 15.68 10	211.28 10.02 10	216.33 19.49 10	205.74 12.41 10
22[a]	Mean SD N	229.99 20.34 10	227.48 8.99 10	236.07 23.08 10	230.56 13.45 10
29[a]	Mean SD N	247.24 22.85 10	243.31 11.44 10	251.03 20.27 10	234.94 9.36 10
36[a]	Mean SD N	264.33 25.31 10	254.00 10.24 10	262.76 19.94 10	249.32 10.30 10
43[a]	Mean SD N	272.55 23.05 10	265.86 11.33 10	266.57 25.33 10	265.42 10.24 10
50[a]	Mean SD N	277.35 22.01 10	270.01 10.31 10	277.73 25.95 10	274.81 14.37 10
57[a]	Mean SD N	288.09 26.97 10	277.04 14.39 10	285.94 26.63 10	283.04 20.46 10
64[a]	Mean SD N	295.48 23.27 10	291.72 12.51 10	299.89 28.84 10	286.18 23.09 10
71[a]	Mean SD N	307.38 25.99 10	291.72 12.51 10	299.89 28.84 10	296.73 23.17 10
78[a]	Mean SD N	310.98 24.68 10	300.19 14.72 10	309.97 24.16 10	304.07 19.27 10
85[a]	Mean SD N	315.01 26.87 10	306.16 13.70 10	311.48 26.33 10	308.49 24.23 10
90[a]	Mean SD N	317.57 27.44 10	306.78 16.76 10	313.61 25.61 10	307.44 24.13 10

Summary of Body Weight Gains (G)

Days relative to start date	Male body weight gain	G1  0 mg/kg/d	G2 100 mg/kg/d	G3  300 mg/kg/d	G4 1000 mg/kg/d
1 - 8 [a]	Mean SD N	61.45 6.30 10	57.43 4.42 10	60.26 3.38 10	55.05* 5.56 10
8-15 [a]	Mean SD N	56.17 10.81 10	59.19 12.11 10	55.79 15.98 10	50.75 12.07 10
15-22[a1]	Mean SD N	35.91 10.81 10	39.38 12.33 10	33.46 5.74 10	34.75 23.53 10
22-29[a1]	Mean SD N	33.36 10.41 10	30.75 6.51 10	29.52 13.59 10	10.85* 21.24 10
29-36[a1]	Mean SD N	25.70 10.65 10	30.09 5.77 10	28.92 4.80 10	12.48 17.82 10
36-43[a]	Mean SD N	32.69 13.79 10	23.34 7.14 10	18.47* 8.19 10	22.02 11.39 10
43-50[a]	Mean SD N	20.07 6.96 10	25.96 8.07 10	24.48 6.55 10	29.67 9.83 10
50-57[a]	Mean SD N	22.08 5.98 10	18.95 7.02 10	13.80 10.03 10	23.29 12.11 10
57-64[a]	Mean SD N	20.70 7.85 10	18.18 7.95 10	17.23 11.06 10	7.46 15.31 10
64-71[a]	Mean SD N	19.74 7.47 10	16.87 8.48 10	14.05 6.11 10	19.30 14.75 10
71-78[a]	Mean SD N	13.85 6.40 10	18.35 9.27 10	15.08 6.40 10	15.21 11.71 10
78-85[a]	Mean SD N	14.57 10.18 10	12.57 9.60 10	9.59 6.24 10	2.29* 11.15 10
85-90[a]	Mean SD N	3.13 7.10 10	7.14 6.67 10	4.07 11.81 10	-10.19 28.95 10

 

Days relative to Start	Female Body Weight Gain	G1 0 mg/kg/d	G2  100 mg/kg/d	G3  300 mg/kg/d	G4 1000 mg/kg/d
1-8[a]	Mean SD N	31.37 5.29 10	29.47 6.07 10	26.95 8.26 10	27.49 5.73 10
8-15[A]	Mean SD N	27.56 6.37 10	25.22 9.05 10	31.97 8.71 10	23.15
15-22 [a]	Mean SD N	15.14 5.53 10	16.20 7.49 10	19.74 10.86 10	24.82 13.33 10
22-29[a]	Mean SD N	17.25 8.08 10	15.83 5.09 10	14.96 9.38 10	4.38* 7.10 10
29-36[a]	Mean SD N	17.09 5.19 10	10.60 4.39 10	11.73 7.14 10	14.38 8.89 10
36-43[a]	Mean SD N	8.22 7.10 10	11.86 6.52 10	3.82 8.94 10	16.10 7.33 10
43-50 [a]	Mean  SD N	4.80 6.48 10	4.14 6.92 10	11.15 7.64 10	9.40 5.97 10
50-57 [a]	Mean SD N	10.74 7.92 10	7.03 11.65 10	8.21 5.52 10	8.22 10.06 10
57-64 [a]	Mean SD N	7.39 6.57 10	9.27 7.88 10	12.84 8.45 10	3.15 8.54 10
64-71 [a]	Mean SD N	11.91 8.11 10	5.41 5.17 10	1.11* 8.31 10	10.54 5.38 10
71-78 [a]	Mean SD N	3.59 6.20 10	8.47 7.86 10	10.08 9.55 10	7.34 5.88 10
78-85 [a]	Mean SD N	4.04 6.74 10	5.98 6.96 10	1.51 6.60 10	4.43 8.01 10
85-90[a]	Mean SD N	2.55 6.61 10	0.62 7.21 10	2.13 5.15 10	-1.06 9.68 10

Summary of Food Consumption

Days relative to start Date	Male Food Consumption	G1 0 mg/kg/d	G2  100 mg/kg/d	G3  300 mg/kg/d	G4 1000 mg/kg/d
1 - 8 [a]	Mean SD N	27.97 1.47 10	27.09 0.91 10	27.91 0.71 10	26.33*
8-15[a]	Mean SD N	30.48 0.79 10	29.16* 0.56 10	29.01* 0.51 10	28.94* 0.95 10
15-22[a]	Mean SD N	29.45 1.29 10	27.03* 1.67 10	26.65* 1.56 10	27.74 1.78 10
22-29 [a]	Mean SD N	27.84 1.84 10	25.61* 1.11 10	24.93* 1.70 10	24.72* 1.77 10
29-36[a]	Mean SD N	29.29 2.03 10	27.51 1.24 10	25.57* 0.92 10	22.22* 4.88 10
36-43 [a]	Mean SD N	30.46 2.20 10	27.83* 2.17 10	28.21 0.78 10	27.18* 0.56 10
43-50[a]	Mean SD N	28.97 1.21 10	27.49* 0.58 10	28.21 0.78 10	27.18* 0.56 10
57-64[a]	Mean SD N	20.70 7.85 10	18.18 7.95 10	17.23 11.06 10	7.46 15.31 10
64-71[a]	Mean SD N	19.74 7.47 10	16.87 8.48 10	14.05 6.11 10	19.30 14.75 10
71-78[a]	Mean SD N	13.85 6.40 10	18.35 9.27 10	15.08 6.40 10	15.21 11.71 10
78-85[a]	Mean SD N	14.57 10.18 10	12.57 9.60 10	9.59 6.24 10	2.29* 11.15 10
85-90[a]	Mean SD N	3.13 7.10 10	7.14 6.67 10	4.07 11.81 10	-10.19 28.95 10

 

Days relative to start Date	Female Food Consumption	G1 0 mg/kg/d	G2  100 mg/kg/d	G3  300 mg/kg/d	G4 1000 mg/kg/d
1 - 8 [a]	Mean SD N	19.55 0.79 10	18.56 1.01 10	18.88 1.07 10	18.68 0.61 10
8-15[a]	Mean SD N	20.00 0.88 10	19.62 1.11 10	19.92 0.47 10	19.38 0.57 10
15-22[a]	Mean SD 10	19.99 1.41 10	18.31* 1.33 10	19.30 1.31 10	20.91 0.99 10
22-29 [a2]	Mean SD N	19.63 1.46 10	19.44 1.08 10	19.15 0.84 10	19.21 1.47 10
29-36[a]	Mean SD N	20.64 1.30 10	19.11 1.37 10	18.72* 1.27 10	19.69 1.91 10
36-43 [a]	Mean SD N	20.87 0.79 10	18.77 0.88 10	18.48* 1.12 10	20.13 1.59 10
43-50[a]	Mean SD N	20.48 0.95 10	19.59 1.06 10	19.29 0.73 10	19.50 0.87 10
57-64[a]	Mean SD N	22.03 0.96 10	19.76* 0.36 10	20.07* 2.11 10	20.95* 1.11 10
64-71[a]	Mean SD N	22.66 1.24 10	19.76* 0.36 10	20.07* 2.11 10	20.95* 1.11 10
71-78[a]	Mean SD N	21.30 4.15 10	19.64 1.29 10	20.03 1.67 10	20.50 0.93 10
78-85[a]	Mean SD N	21.98 0.70 10	19.80* 1.13 10	18.91 0.99 10	19.81* 1.00 10
85-90[a]	Mean SD N	22.90 2.38 10	18.41* 1.36 10	18.54* 1.36 10	19.16* 1.54 10

Clinical Signs - Full tables can be found in Appendix 1 of attached study report, page 109.

	Males				Females
Observations from day 1 to 91	G1	G2	G3	G4	G1	G2	G3	G4
Normal	10	10	10	10	10	10	10	10
Dehydration	0	0	0	1	0	0	0	0
Salivation	0	0	10	10	0	0	10	10

Summary of Opthalmological Findings- Full tables can be found in Appendix 3 of attached full study report, table 121.

	Males				Females
Observations from day 1 to 91	G1	G2	G3	G4	G1	G2	G3	G4
Normal	10	10	10	10	10	10	10	10

Functional Observational Battery- Full tables can be found in Appendix 6 of the full study report attached, page 137.

	Males				Females
Observations from day 1 to 91	G1	G2	G3	G4	G1	G2	G3	G4
Neurobehavioural
Posture
Sitting Normally, feet tucked	0	1	0	0
Sitting or Standing Alert	10	9	10	10	9	6	6	5
Abnormal Vocalizations abscent	10	10	10	10	10	10	10	10
Tremours abscent	10	10	10	10	10	10	10	10
Convulsions, clonic movement or tonic movement abscent	10	10	10	10	10	10	10	10
Ease of removing from cage (no aggression)	10	10	10	10	10	10	10	10
Low handling reactivity	10	10	10	10	10	10	10	10
Eyes wide open, normal	10	10	10	10	10	10	10	10
Eye examination normal	10	10	10	10	10	10	10	10
Abscent of piloerection	10	10	10	10	10	10	10	10
Abscent of lacrimation	10	10	10	10	10	10	10	10
Salivation, none	10	10	10	10	10	10	10	10
Skin/Fur normal	10	10	10	10	10	10	10	10
Perineum wetness abscent	10	10	10	10	10	10	10	10
Respiration normal	10	10	10	10	10	10	10	10
Muscle Tone normal	10	10	10	10	10	10	10	10
Extensor thrust response normal	10	10	10	10	10	10	10	10
Open Feild Observations
Gait normal	10	10	10	10	10	10	10	10
Tremours	10	10	10	10	10	10	10	10
No impaired mobility	10	10	10	10	10	10	10	10
Arousal level Alert	10	10	10	10	10	10	10	10
Stereotypic behavour abscent	10	10	10	10	10	10	10	10
Bizarre behavour	10	10	10	10	10	10	10	10
Urination Abscent Normal	8 2	7 3	8 2	8 2	7 3	8 2	8 2	8 2
Defecation Abscent Normal	4 6	5 5	5 5	7 3	9 1	8 2	8 2	8 2
Abnormal vocalization abscent	10	10	10	10	10	10	10	10
Sensory Reactivity Measurements
Approach response normal	10	10	10	10	10	10	10	10
Touch response normal	10	10	10	10	10	10	10	10
Click Response	10	10	10	10	10	10	10	10
Tail pinch response normal	10	10	10	10	10	10	10	10
Pupil response normal	10	10	10	10	10	10	10	10
Aerial righting reflex normal	10	10	10	10	10	10	10	10
Hindlimb footsplay (mm) Mean SD	82.67 7.61	84.67 8.88	89.37 12.80	75.13 9.31	54.40 7.12	58.87 7.14	55.53 5.79	50.30 6.37
Gripstrength (gf) Average SD	814.17 36.65	847.20 43.01	921.43* 44.97	844.47 43.08	558.00 23.57	645.63 18.68	636.53 24.62	643.60 18.12

Summary of Estrous Cycle Prior to Necropsy- Full tables can be found in Appendix 14 of the full study report attached, page 185.

Stage of Estrous	Days Relative to Start Date		G1 0 mg/kg/d	G2 100 mg/kg/d	G3  300 mg/kg/d	G4 1000 mg/kg/d
D	91	N+ve	6	1	6	4
E	91	N+ve	0	2	2	4
M	91	N+ve	0	0	0	0
P	91	N+ve	4	7	2	2

Haematology and Coagulation Parameters- Full tables can be found in the pathology report within the full study report attached, page 217.

Parameter	Days Relative to start date		Males G1 0 mg/kg/d	Males G2 100  mg/kg/d	Males G3  300 mg/kg/d	Males G4 1000 mg/kg/d	Females G1 0 mg/kg/d	Females G2 100 mg/kg/d	Females G3  300 mg/kg/d	Females G4 1000 mg/kg/d
RBC (10^12/L)	91[a]	Mean SD N %Dif	9.10 0.21 10 -	8.79 0.36 10 -3.41	8.86 0.26 10 -2.57	8.95 0.49 10 -1.57	8.13 0.37 10 -	8.18 0.24 10 0.63	8.33 0.24 10 2.43	8.14 0.38 10 0.04
HGB (g/L)	91[a]	Mean SD N %dif	162 4 10 -	157* 4 10 -3	160 4 10 -1	163 4 10 1	154 6 10 -	152 6 10 -1	157 7 10 2	151 5 10 -2
HCT (L/L)	91[a1]	Mean SD N %dif	0.489 0.012 10 -	0.478 0.022 10 -2.310	0.477 0.016 10 -2.413	0.494 0.023 10 1.063	0.465 0.025 10-	0.464 0.016 10 -0.215	0.478 0.022 10 2.882	0.465 0.015 10 -0.065
MCV (fL)	91 [a]	Mean SD N %Dif	53.8 1.6 10 -	54.4 1.9 10 1.2	53.9 1.6 10 0.2	55.3 1.6 10 2.8	57.2 1.6 10 -	56.7 1.8 10 -0.8	57.4 1.6 10 0.4	57.2 1.0 10 0.1
MCH (pg)	91[a]	Mean SD N %Dif	17.8 0.6 10 -	17.9 0.5 10 0.3	18.1 0.6 10 1.4	18.2 0.8 10 2.5	19.0 0.4 10 -	18.6 0.5 10 -2.0	18.8 0.5 10 -0.6	18.5 0.5 10 -2.2
MCHC (g/L)	91 [a]	Mean SD N %Dif	331 7 10 -	328 10 10 -1	335 12 10 1	330 11 10 0	331 9 10 -	327 7 10 -1	328 4 10 -1	324 7 10 -2
Retic A (10^12/L)	91[a]	Mean SD N %dif	0.155 0.023 10 -	0.161 0.025 10 3.542	0.135 0.033 10 -13.200	0.129 0.038 10 -16.742	0.163 0.019 10 -	0.160 0.021 10 -1.782	0.166 0.031 10 2.090	0.176 0.047 10 7.929
PLT  (10^9/L)	91[a]	Mean SD N %dif	1736 185 10 -	1826 137 10 5	1980* 83 10 14	2115* 193 10 22	1771 184 10 -	1912 99 10 8	2107* 168 10 19	2029* 203 10 15
WBC (10^9/L)	91[a]	Mean SD N %Dif	10.03 2.04 10 -	9.77 1.80 10 -2.61	10.20 1.56 10 1.68	11.53 3.24 10 14.89	5.64 1.43 10 -	5.85 2.16 10 3.63	6.19 2.11 10 9.59	7.48 2.71 10 32.49
Neut A (10^9/L)	91[a1]	Mean SD N %dif	2.46 0.44 10 -	2.65 1.04 10 7.89	2.62 0.59 10 6.63	3.67 1.79 10 49.27	1.23 0.38 10 -	1.42 0.82 10 14.92	1.50 0.61 10 21.90	2.47* 1.31 10 100.49
Lymp A (10^9/L)	91[a]	Mean SD N %dif	6.84 1.88 10 -	6.46 1.31 10 -5.60	6.86 1.05 10 0.29	7.09 1.88 10 3.66	4.04 1.49 10 -	4.04 1.67 10 0.07	4.27 1.40 10 5.82	4.58 1.69 10 13.27
Mono A (10^9/L)	91[a2]	Mean SD N %Dif	0.27 0.11 10 -	0.27 0.09 10 0.37	0.30 0.12 10 8.86	0.41 0.22 10 49.82	0.13 0.04 10 -	0.15 0.08 10 14.17	0.14 0.08 10 12.60	0.15 0.06 10 21.26
Baso A (10^9/L)	91[a]	Mean SD N %Dif	0.03 0.01 10 -	0.02 0.01 10 -17.86	0.02 0.01 10 -17.86	0.03 0.02 10 7.14	0.01 0.01 10 -	0.01 0.01 10 37.50	0.01 0.01 10 37.50	0.01 0.01 10 62.50
Eosi A (10^9/L)	91 [a2]	Mean SD N %Dif	0.16 0.06 10 -	0.17 0.06 10 7.55	0.26 0.13 10 62.26	0.19 0.15 10 21.38	0.15 0.11 10 -	0.09 0.07 10 -38.41	0.10 0.03 10 -33.77	0.17 0.09 10 9.93
Prothrombin Time (seconds)	91[a]	Mean SD N %Dif	20.1 1.3 10 -	20.0 1.4 10 -0.5	21.1 1.7 10 4.9	19.7 1.3 10 -2.0	19.7 1.4 10 -	19.6 1.6 10 -0.6	19.8 1.1 10 0.2	20.7 1.7 10 5.1
APTT (seconds)	91[a1]	Mean SD N %Dif	7.9 1.6 10 -	7.6 1.7 10 -3.8	7.8 0.8 10 -1.9	8.0 1.0 10 1.4	7.9 1.6 10 -	8.9 1.1 10 13.4	7.3 0.8 10 -7.1	7.8 1.7 10 -0.4

 

Summary of Clininal Chemistry- Full tables can be found in the pathology report within the full study report attached, page 229

Parameter	Days Relative to Start Date		Male G1 0 mg/kg/d	Males G2 100 mg/kg/d	Males G3 300 mg/kg/d	Males G4 1000 mg/kd/d	Female G1 0 mg/kg/d	Female G2 100 mg/kg/d	Female G3 300 mg/kg/d	Female G4 1000 mg/kg/d
Glu (mmol/L)	91[1]	Mean SD N %Dif	7.10 0.70 10 -	7.33 0.90 10 3.27	7.01 0.65 10 -1.23	6.48 0.97 10 -8.69	7.24 0.89 10 -	6.97 0.51 10 -3.82	6.69 1.01 10 -7.59	6.76 0.59 10 -6.68
BUN (mmol/L)	91[a1]	Mean SD N %Dif	4.83 0.61 10 -	3.97* 0.51 10 -17.98	4.20 0.63 10 -13.14	5.43 1.81 10 12.37	5.42 1.13 10 -	4.63 0.38 10 -14.56	5.41 0.44 10 -0.28	4.55 1.46 10 -16.06
Creat (umol/L)	91[a]	Mean SD N %Dif	34 7 10 -	36 6 10 6	19 6 10 -14	33 9 10 -3	40 6 10 -	39 7 10 -3	37 5 10 -7	32* 5 10 -20
AST (U/L)	91[a]	Mean SD N %Dif	101 23 10 -	104 28 10 2	126 23 10 25	144* 31 10 42	106 24 10 -	105 30 10 -1	157* 55 10 49	135 35 10 28
ALT (U/L)	91[a1]	Mean SD N %Dif	44 15 10 -	50 14 10 12	158* 71 10 257	242* 124 10 445	34 7 10 -	34 16 10 0	113* 97 10 231	201* 118 10 485
Alp (U/L)	91[a]	Mean SD N %Dif	76 11 10 -	69 15 12 -9	73 12 10 -4	46* 18 10 -39	49 10 10 -	39* 5 10 -20	44 10 10 -10	29* 7 10 -42
T.Bil (umol/L)	91[a2]	Mean SD N %Dif	2.33 0.81 10 -	1.87 0.56 10 -19.90	1.95 0.72 8b -16.38	1.72 0.93 8b -26.14	2.05 0.71 9b -	2.00 0.72 10 -2.40	1.94 0.59 10 -5.27	2.07 0.57 9b 1.08
T.Chol (mmol/L)	91[a]	Mean SD N %Dif	2.07 0.33 10 -	2.23 0.26 10 7.62	1.92 0.25 10 -7.33	1.63* 0.33 10 -21.37	2.36 0.33 10 -	2.23 0.26 10 -5.75	2.23 0.27 10 -5.63	1.61* 0.34 10 -31.73
HDL cholesterol (mmol/L)	91[a]	Mean SD N %Dif	1.69 0.29 10 -	1.82 0.25 10 7.39	1.52 0.22 10 -10.23	1.24* 0.29 10 -26.61	1.78 0.30 10 -	1.78 0.23 10 0.06	1.74 0.23 10 -2.30	1.19* 0.27 10 -33.43
LDL  cholesterol (mmol/L)	91[a]	Mean SD N %Dif	0.17 0.11 10 -	0.29* 0.06 10 76.02	0.25 0.09 10 52.28	0.29* 0.11 10 71.34	0.44 0.14 10 -	0.36 0.07 10 -19.05	0.40 0.13 10 -9.75	0.34 0.09 10 -24.24
Trig (mmol/L)	91[a1]	Mean SD N %Dif	1.08 0.46 10 -	0.61* 0.23 10 -43.59	0.75 0.24 10 -30.76	0.52* 0.17 10 -52.04	0.71 0.30 10 -	0.44* 0.10 10 -37.30	0.46* 0.16 10 -34.61	0.47* 0.17 10 -33.76
T.Pro (g/L)	91[a]	Mean SD N %Dif	68.6 2.4 10 -	67.8 2.4 10 -1.2	65.6* 2.0 10 -4.3	61.5* 2.6 10 -10.3	68.2 1.8 10 -	68.2 3.1 10 -0.1	65.8 3.3 10 -3.6	64.8* 2.4 10 -5.0
ALB (g/L)	91[a]	Mean SD N %Dif	33.9 1.6 10 -	32.9 1.3 10 -2.9	32.8 1.2 10 -3.3	31.1* 1.9 10 -8.1	36.2 2.3 10 -	35.8 1.5 10 -1.2	34.1 2.4 10 -5.9	33.8* 2.0 10 -6.8
GLOB (g/L)	91[a]	Mean SD N %Dif	34.7 1.4 10 -	34.9 1.5 10 0.5	32.9* 1.1 10 -5.2	30.4* 2.0 10 -12.5	32.0 1.8 10 -	32.4 2.1 10 1.2	31.7 1.9 10 -1.0	31.0 3.0 10 -3.0
A/G (ratio)	91[a]	Mean SD N %Dif	0.98 0.05 10 -	0.94 0.05 10 -3.41	1.00 0.03 10 1.97	1.03 0.10 10 5.31	1.14 0.12 10 -	1.11 0.07 10 -2.65	1.08 0.10 10 -5.13	1.10 0.14 10 -3.42
Pi (mmol/L)	91[a1]	Mean SD N %Dif	1.86 0.21 10 -	1.72 0.18 10 -7.44	1.81 0.13 10 -2.64	1.70 0.24 10 -8.35	1.59 0.20 10 -	1.46 0.22 10 -8.59	1.54 0.28 10 -3.26	1.58 0.15 10 -1.13
Ca (mmol/L)	91[a1]	Mean SD N %Dif	2.82 0.10 10 -	2.89 0.09 10 2.66	2.95* 0.08 10 4.62	2.90 0.11 10 2.95	2.86 0.06 10 -	2.96* 0.10 10 3.46	2.89 0.07 10 1.22	2.93 0.10 10 2.55
Na (mEq/L)	91[a]	Mean SD N %Dif	148.5 1.7 10 -	148.6 1.4 10 0.1	148.3 1.2 10 -0.1	146.9 3.4 10 -1.1	146.5 1.5 10 -	146.2 1.4 10 -0.2	146.2 1.6 10 -0.2	144.0* 1.4 10 -1.7
K (mEq/L)	91[a1]	Mean SD N %Dif	3.75 0.27 10 -	3.83 0.34 10 2.11	3.98 0.39 10 3.13	3.39 0.61 10 4.66	3.54 0.19 10 -	3.33 0.30 10 -6.04	3.47 0.24 10 -1.89	3.48 0.32 10 -1.61
Cl (mEq/L)	91[a]	Mean SD N %Dif	95.5 1.0 10 -	96.0 0.9 10 0.5	96.5 1.1 10 1.0	95.0 2.2 10 -0.5	95.3 1.0 10 -	95.0 1.0 10 -0.4	95.1 0.9 10 -0.2	93.9 2.0 10 -1.5

b Values below LLOQ were not considered for analysis

[a2] Anova & Dummet (log) Statistically significant difference from control at p <0.05

 

Summary of Thyroid Hormone Profile Day 91- Full tables can be found in the pathology report within the full study report attached, page 241.

			Male G1 0 mg/kg/d	Males G2 100 mg/kg/d	Males G3 300 mg/kg/d	Males G4 1000 mg/kd/d	Female G1 0 mg/kg/d	Female G2 100 mg/kg/d	Female G3 300 mg/kg/d	Female G4 1000 mg/kg/d
TSH(ng/mL)	91[a]	Mean SD N %Dif	1.75 1.21 10 -	1.77 1.08 10 0.91	1.95 1.08 10 11.53	1.69 0.85 10 -3.71	0.86 0.47 9a -	0.60 0.35 10 -30.27	0.84 0.49 10 -3.28	1.22 0.95 10 41.31
T3 (ng/mL)	91[a1]	Mean SD N %Dif	0.39 0.05 9a -	0.46 0.14 9a 18.97	0.43 0.07 9a 12.36	0.39 0.07 10 0.09	0.30 0.08 8a -	0.33 0.06 9a 7.91	0.33 0.09 9a 7.18	0.33 0.07 8a 7.00
T4  (ng/mL)	91[a]	Mean SD N %Dif	33.85 9.05 10 -	25.78 7.75 10 -23.85	29.39 7.63 10 -13.19	26.05 9.29 10 -23.04	11.62 4.51 10 -	15.33 6.33 10 31.93	13.69 3.15 10 17.86	15.89* 4.88 10 36.80

Summary of Fasting Body weight and Organ Weights- Full tables available in the pathology report within the full study report attached, page 249.

Parameter	Days relative to start date		Male G1 0 mg/kg/d	Males G2 100 mg/kg/d	Males G3 300 mg/kg/d	Males G4 1000 mg/kd/d	Female G1 0 mg/kg/d	Female G2 100 mg/kg/d	Female G3 300 mg/kg/d	Female G4 1000 mg/kg/d
Terminal Fasting BW (g)	91[a]	Mean SD N %Dif	526.05 59.38 10 -	524.36 38.76 10 -0.32	492.54 47.06 10 -6.37	431.48* 72.36 10 -17.98	301.55 22.65 10 -	291.27 13.43 10 -3.41	295.88 24.27 10 -1.88	289.01 23.03 10 -4.16
Adrenals (g)	91[a]	Mean SD N %Dif	0.0722 0.0123 10 -	0.0802 0.0111 10 11.0695	0.0722 0.0106 10 0.000	0.0932* 0.0146 10 29.1493	0.0952 0.0167 10 -	0.0895 0.0163 10 -5.9065	0.0986 0.0131 10 3.6469	0.0981 0.0113 10 3.0793
Brain (g)	91[a]	Mean SD N %Dif	2.1952 0.1132 10 -	2.2086 0.0745 10 0.6077	2.1677 0.0915 10 -1.2568	2.1230 0.1563 10 -3.2894	2.0488 0.0713 10 -	1.9782 0.0538 10 -3.4435	2.0235 0.0545 10 -1.2339	1.9833 0.0892 10 -3.1971
Epididymides (g)	91[a]	Mean SD N %Dif	1.4920 0.2297 10 -	1.6843 0.1759 10 12.8900	1.6487 0.1257 10 10.5026	1.4146 0.2155 10 -5.1890
Heart (g)	91[a]	Mean SD N %Dif	1.4451 0.1902 10 -	1.4477 0.1486 10 0.1765	1.4055 0.2369 10 -2.7424	1.2481* 0.1548 10 -13.6363	0.9714 0.0911 10 -	1.0037 0.0678 10 3.3210	1.0339 0.0845 10 6.4382	0.9853 0.1274 10 1.4351
Kidneys (g)	91[a]	Mean SD N %Dif	3.0039 0.3019 10 -	2.9366 0.2104 10 -2.2401	2.6799 0.4067 10 -10.7866	2.6491 0.4940 10 -11.8100	1.6889 0.2136 10 -	1.6643 0.1259 10 -1.4560	1.6765 0.1393 10 -0.7372	1.8063 0.1296 10 6.9517
Liver (g)	91[a]	Mean SD N %Dif	13.8844 1.6854 10 -	14.0126 1.3151 10 0.9236	12.7508 1.9720 10 -8.1649	10.8761* 1.8516 10 -21.6669	7.8564 0.8942 10 -	8.1197 0.5052 10 3.3504	8.1644 0.9202 10 3.9196	8.3569 0.5676 10 6.3706
Pituitary (g)	94 [a] Weighed after fixation	Mean SD N %Dif	0.0145 0.0022 10 -	0.0140 0.0012 10 -3.4459	0.0133 0.0022 10 -8.0634	0.0123 0.0017 10 -15.2309	0.0152 0.0021 10 -	0.0142 0.0030 10 -6.7017	0.0155 0.0021 10 1.9054	0.0141 0.0022 10
Prostate (g)	91[a]	Mean SD N %Dif	1.1757 0.1294 10 -	1.1292 0.2383 10 -3.9541	1.0145 0.1496 10 -13.7166	0.9226* 0.2319 10 -21.5262
Ovaries (g)	91[a]	Mean SD N %Dif					0.1115 0.0188 10 -	0.1208 0.0206 10 8.3027	0.1020 0.0129 10 -8.5089	0.1182 0.0198 10 5.9715
Seminal vesicles & coagulating glands (g)	91[a1]	Mean SD N %Dif	2.0870 0.1227 10 -	2.2751 0.2389 10 9.0145	2.0837 0.2273 10 -0.1567	1.7315 0.5612 10 -17.0347
Spleen (g)	91 [a2]	Mean SD N %Dif	1.0112 0.1468 10 -	1.0105 0.1042 10 -0.0761	0.9246 0.1842 10 -8.5687	0.8279* 0.1885 10 -18.1302	0.6629 0.0914 10 -	0.6796 0.1203 10 2.5254	0.6969 0.0470 10 5.1414	0.6725 0.0859 10 1.4573
Testes (g)	91[a1]	Mean SD N %Dif	3.5209 0.7312 10 -	4.0162 0.3695 10 14.0667	3.8458 0.2610 10 9.2271	3.4749 0.6165 10 -1.3068
Thymus (g)	91[a]	Mean SD N %Dif	0.4508 0.1077 10 -	0.4110 0.0961 10 -8.8227	0.3618 0.0672 10 -19.7329	0.2882* 0.1336 10 -36.0605	0.4150 0.0815 10 -	0.3751 0.0840 10 -9.6178	0.4042 0.0720 10 -2.5906	0.3527 0.0741 10 -14.9966
Thyroid with parathyroids	94 [a] Weighed after fixation	Mean SD N %Dif	0.372 0.0056 10 -	0.0376 0.0040 10 1.0215	0.0389 0.0061 10 4.5699	0.0341 0.0047 10 -8.3333	0.0311 0.0044 10 -	0.297 0.0058 10 -4.7543	0.0337 0.0036 10 8.2878	0.0297 0.0038 10 -4.4973
Uterus with cervix (g)	91[a]	Mean SD N %Dif					0.6614 0.1585 10 -	0.6135 0.0844 10 -7.2482	0.7566 0.2191 10 14.3980	0.9141 0.4469 10 38.2108
Body weight to Organ Ratios
Adrenals (%)	91[a1]	Mean SD N %Dif	0.0138 0.0024 10 -	0.0153 0.0022 10 10.9501	0.0147 0.0023 10 6.6657	0.0222* 0.0057 10 60.4052	0.0315 0.0046 10 -	0.0308 0.0061 10 -2.1511	0.0334 0.0042 10 5.9711	0.0342 0.0052 10 8.4136
Brain (%)	91[a]	Mean SD N %Dif	0.4207 0.0363 10 -	0.4235 0.0374 10 0.6582	0.4434 0.0420 10 5.3918	0.5010* 0.0638 10 19.0880	0.6823 0.0481 10 -	0.6803 0.0315 10 -0.2960	0.6872 0.0469 10 0.7204	0.6899 0.0615 10 1.1190
Epididymides (%)	91[a2]	Mean SD N %Dif	0.2854 0.0428 10 -	0.3215 0.0285 10 12.6378	0.3356* 0.0156 10 17.5685	0.3350 0.0677 10 17.3755
Heart (%)	91[a1]	Mean SD N %Dif	0.2752 0.0259 10 -	0.2759 0.0163 10 0.2352	0.2866 0.0285 10 4.1332	0.2928 0.0343 10 6.3984	0.3224 0.0228 10 -	0.3447 0.0198 10 6.9055	0.3514 0.0380 10 8.9908	0.3404 0.0301 10 5.5795
Kidneys (%)	91[a1]	Mean SD N %Dif	0.5731 0.0418 10 -	0.5619 0.0464 10 -1.9574	0.5440 0.0610 10 -5.0811	0.6171 0.0824 10 7.6769	0.5597 0.0519 10 -	0.5717 0.0397 10 2.1506	0.5694 0.0577 10 1.7398	0.6269* 0.0456 10 12.0099
Liver (%)	91[a]	Mean SD N %Dif	2.6385 0.1282 10 -	2.6740 0.1748 10 1.3467	2.5776 0.2029 10 -2.3079	2.5280 0.1781 10 -4.1872	2.6031 0.1741 10 -	2.7911 0.1825 10 7.2225	2.7593 0.2082 10 6.0033	2.9016* 0.2336 10 11.4695
Pituitary (%)	94b [a]	Mean SD N %Dif	0.0028 0.0004 10 -	0.0027 0.0002 10 -3.7007	0.0027 0.0003 10 -3.0629	0.0029 0.0004 10 3.8445	0.0051 0.0006 10 -	0.0049 0.0010 10 -3.3264	0.0053 0.0007 10 4.1114	0.0049 0.0007 10 -3.1177
Prostate (%)	91[a1]	Mean SD N %Dif	0.2275 0.0475 10 -	0.2166 0.0490 10 -4.7974	0.2064 0.0261 10 -9.2782	0.2139 0.0341 10 -6.0129
Seminal vesicles & coagulating glands (%)	91[a]	Mean SD N %Dif	0.4018 0.0548 10 -	0.4346 0.0432 10 8.1606	0.4242 0.0399 10 5.5631	0.3962 0.0918 10 -1.4005
Spleen (%)	91[a]	Mean SD N %Dif	0.1928 0.0220 10 -	0.1930 0.0173 10 0.134	0.1871 0.0257 10 -2.9705	0.1916 0.0250 10 -0.6056	0.2198 0.0230 10 -	0.2328 0.0358 10 5.9301	0.2365 0.0198 10 7.6386	0.2325 0.0228 10 5.8084
Testes (%)	91[a2]	Mean SD N %Dif	0.6757 0.1478 10 -	0.7690 0.0826 10 13.7989	0.7858 0.0751 10 16.2852	0.8329 0.2241 10 23.2595
Thymus(%)	91[a]	Mean SD N %Dif	0.0850 0.0132 10 -	0.0779 0.0140 10 -8.3537	0.0736 0.0126 10 -13.4802	0.0641* 0.0222 10 -24.6412	0.1376 0.0254 10 -	0.1295 0.0316 10 -5.9071	0.1365 0.0212 10 -0.8353	0.1222 0.0253 10 -11.2068
Thyroid with parathyroids (%)	94b[a]	Mean SD N %Dif	0.0071 0.0011 10 -	0.0072 0.0005 10 0.7159	0.0079 0.0011 10 11.3076	0.0081 0.0015 10 13.3128	0.0103 0.0013 10 -	0.0102 0.0019 10 -1.7220	0.0114 0.0014 10 10.6795	0.0103 0.0011 10 -0.3650
Brain (g)	91[a]	Mean SD N %Dif	2.1952 0.1132 10 -	2.2086 0.0745 10 0.6077	2.1677 0.0915 10 -1.2568	2.1230 0.1563 10 -3.2894	2.0488 0.0713 10 -	1.9782 0.0538 10 -3.4435	2.0235 0.0545 10 -1.2339	1.9833 0.0892 10 -3.1971
Adrenals (%)	91[a]	Mean SD N %Dif	3.2863 0.5134 10 -	3.6329 0.5153 10 13.5457	3.3399 0.5470 10 1.6297		4.6520 0.8654 10 -	4.5375 0.8949 10 -2.4610	4.8659 0.5740 10 4.5974	4.9563 0.6303 10 6.5412
Epididymides (%)	91[a]	Mean SD N %Dif	68.0096 9.9367 10 -	76.3642 8.4878 10 12.2844	76.0880 5.3290 10 11.8783	66.6391 8.8492 10 -2.0151
Heart (%)	91[a]	Mean SD N %Dif	65.7410 7.3030 10 -	65.6399 7.2554 10 -0.1538	64.8224 5.0801 10 -1.3973	58.8222 6.1158 10 -10.5243	47.4024 3.9913 10 -	50.7558 3.4969 10 7.0743	51.1232 4.2533 10 7.8494	49.6844 5.9841 10 4.8140
Kidneys (%)	91[a]	Mean SD N %Dif	136.7620 11.2261 10 -	133.1287 10.8318 10 -2.6567	124.0262 20.9710 10 -9.3124	124.0779 16.0747 10 -9.2746	82.3550 9.2292 10 -	84.2156 7.0193 10 2.2592	82.8493 6.4089 10 0.6003	91.2506* 7.8767 10 10.8016
Liver(%)	91[a]	Mean SD N %Dif	630.9917 56.6984 10 -	634.5829 56.1159 10 0.5691	588.9430 95.0693 10 -6.6639	510.9549* 68.4430 10 -19.0235	383.4500 40.4778 10 -	410.6466 26.8349 10 7.0926	403.1863 41.4176 10 5.1470	422.2106 34.2184 10 10.1084
Pituitary (%)	94b [a]	Mean SD N %Dif	0.6599 0.0809 10 -	0.6341 0.0475 10 -3.8995	0.6165 0.1055 10 -6.5685	0.5802 0.0764 10 -12.0760	0.7437 0.1057 10 -	0.7173 0.1465 10 -3.5558	0.7657 0.0972 10 2.9469	0.7124 0.1050 10 -4.2128
Prostate (%)	91[a]	Mean SD N %Dif	53.7753 7.3737 10 -	51.0966 10.4659 10 -4.9813	46.8193 6.7512 10 -12.9353	43.3795 10.5331 10 -19.3320
Seminal vesicles & coagulating glands (%)	91[a]	Mean SD N %Dif	95.3654 8.4421 10 -	103.2154 12.3273 10 8.2316	96.2262 11.0214 10 0.9027	80.8311 23.7675 10 -15.2407
Spleen (%)	91[a1]	Mean SD N %Dif	46.0378 5.8582 10 -	45.7742 4.6183 10 -0.5726	42.7822 9.1872 10 -7.0717	38.7747* 7.1841 10 -15.7764	32.3085 3.8053 10 -	34.3185 5.7734 10 6.2212	34.4703 2.5705 10 6.6912	33.9758 4.6370 10 5.1607
Testes (%)	91[a1]	Mean SD N %Dif	160.8431 33.9820 10 -	182.2903 20.8059 10 13.3342	177.5650 12.0894 10 10.3964	164.4953 30.9099 10 2.2707
Thymus (%)	91[a]	Mean SD N %Dif	20.4097 4.2144 10 -	18.6367 4.4453 10 -8.6867	16.6408 2.6943 10 -18.4660	13.3151* 5.7203 10 -34.7605	20.2379 3.8161 10 -	18.9827 4.3206 10 -6.2025	19.9914 3.6565 10 -1.2184	17.8219 3.8897 10 -11.9384
Thyroid with parathyroids (%)	94[a]   Weighed after fixation	Mean SD N %Dif	1.6938 0.2330 10 -	1.7030 0.1910 10 0.5464	1.7952 0.2662 10 5.9889	1.6151 0.2758 10 -4.6453	1.5202 0.2125 10 -	0.4971 0.2797 10 -1.5171	1.6648 0.1608 10 9.5140	1.4990 0.1779 10 -1.3914
Uterus with Cervix	91[a1]	Mean SD N %Dif					32.2635 7.6291 10 -	31.0489 4.5465 10 -3.7645	37.4834 11.3409 10 16.1789	46.0266 22.6973 10 42.6585
Ovaries	91 [a1]	Mean SD N %Dif					5.4445 0.8986 10 -	6.1101 1.0788 10 12.2256	5.0511 0.7050 10 -7.2250	5.9701 1.0422 10 9.6549

Additional Tabulated data is available in the Pathology Report, affixed in appendix 15 of the attached fully study report, including full details of histopathology, urinology and statistical treatment of results. 

 

An assesment of the stages of spermatogenesis was reported in Appendix 33 of the pathology report. All stages were within normal limits for all treatment and control groups. 

Applicant's summary and conclusion

Conclusions:

In conclusion, oral gavage administration of Reaction product of fatty acids, C18 alkyl with amines, polyethylenepoly-tetraethylenepentamine fraction at 100, 300 and 1000 mg/kg/day doses to Wistar rats for 90 consecutive days didnot result in any treatment-related clinical signs or mortality. The treatment decreased the body weights in males at 1000 mg/kg/day, associated with lower food consumption.

Clinical pathology investigation revealed test item related increase in total leukocyte count, neutrophils and monocytes in both males and females at 1000 mg/kg/day correlated with increased extramedullary hematopoiesis in spleen and adrenals and increased cellularity in bone marrow (sternum and femur). Test iteam related decrease in total protein/albumin/globulin, triglycerides, cholesterol and AHDL were noted in both males and females at 1000 mg/kg/day. All these changes were completely reversible except for the reduced protein profile in females at the end of recovery period. All the changes in leukocytes parameters and protein profile were secondary to the local inflammatory condition (mucosal necrosis) noted in the non-glandular stomach and these systemic or local changes were considered non-adverse. Increased ALT level noticed in both sex at ≥ 300 mg/kg/day without any correlating microscopic lesions in liver was considered as non-adverse effect of the test item.

The decreased terminal fasting body weight was noticed in males (18%) at 1000 mg/kg/day and was reversible. At 1000 mg/kg/day, increased adrenal weights (microscopically, associated with increased extramedullary hematopoiesis); decreased thymus weights (grossly, small and microscopically, associated with decreased cellularity-cortex/medulla in few males and females) and decreased spleen (microscopically, decreased cellularity-marginal zone of white pulp in two males) and prostate weights (microscopically, atrophy withdecreased secretion in a male) were noticed. Reductions in weight of spleen, thymus, heart and prostate and increased brain weights in males at1000 mg/kg/day were considered secondary effects due to either stress due to high dose levels used, decreased feed intake and/or reduced terminal body weight. The reduced thymus weight was also noted in males at 300 mg/kg/day without any histological correlation. These organ weight changes were not considered as adverse.
Grossly, thickening of non-glandular mucosa (stomach) at 1000 mg/kg/day

males and females was histologically accompanied by epithelial hyperplasia/ mucosal necrosis. The range of microscopic changes were suggestive of forestomach gastritis specifically observed in rodents and was attributed to possible irritant potential of the test item.

Executive summary:

The purpose of this repeated dose toxicity study (OECD guideline 408) was to evaluate the systemic toxicity profile of the test item, Reaction product of fatty acids, C18 alkyl with amines, polyethylenepoly-tetraethylenepentamine fraction, in Wistar rats when administered orally by gavage for 90 consecutive days and to assess the reversibility of any effects during a subsequent 28 days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL).

The test item was weighed and suspended in vehicle, i.e., Corn oil and administered to rats at the graduated dose levels of 100, 300 and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4 ) / high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/ vehicle control recovery (G1R) groups received vehicle Corn oil alone. The dose volume administered was 5 mL/kg body weight. Each main group in the experiment was comprised of 10 male and 10 female rats and recovery groups comprised of 5 male and 5 female rats.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not performed at the test facility. The stability and homogeneity of the test item in the vehicle were established at 5 and 500 mg/mL concentrations under Eurofins Advinus Study No. G18456. Based on the results, the test item was stable and homogenous in the vehicle up to 3 days when stored at room temperature.

During the conduct of this study, the prepared dose formulations and vehicle (corn oil) were analyzed for homogeneity and active ingredient (a.i.) concentration on test Day 1 and during 2nd (Day 35) and 3rd (Day 69) month of the treatment. The results indicated that the percent agreement of the analyzed concentrations were in the range, 80% to 120% of the claimed concentrations and the overall % RSD from six replicates at each dose level were <=15%. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups. The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination. Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.

All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratios were derived as percent fasting body weights and brain weight. Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose group animals (G4) including all gross lesions. The testes sections were also examined for the qualitative assessment of stages of spermatogenesis. Further, following organs suspected of showing test item-related histopathological changes in high dose (G4) group were examined in the respective lower dose groups and recovery groups.

- Males- Liver, stomach, mesenteric lymph nodes, spleen, thymus, adrenals, testes, epididymides, seminal vesicles, coagulating gland, prostate, sternum with marrow, femur bone and bone marrow smears.

- Females- Liver, stomach, mesenteric lymph nodes, spleen, thymus, adrenals, sternum with marrow, femur bone and bone marrow smears.

Under the experimental conditions employed, the following results were obtained:

• Clinical Signs and Mortality: Transient clinical sign of slight salivation was observed soon after the dose administration in all animals at 1000 mg/kg bwt/day starting from treatment Day 5 and at 300 mg/kg bwt/day starting from treatment Day 45. This finding may be attributed to oral mucosa irritation and of no toxicological significance.

• Ophthalmological Examination: Ophthalmological examination did not reveal any ocular abnormalities.

• Neurological Findings: No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.

• Body Weights and Food Consumption: The treatment decreased the body weights in males at 1000 mg/kg/day, associated with lower food consumption in males. The body weights were unaffected at 100 and 300 mg/kg/day in males and at all the doses tested in females.

• Clinical Pathology Investigation: Test item related increase in total leukocyte count, neutrophils and monocytes in both males and females at 1000 mg/kg/day correlated with increased extramedullary hematopoiesis in spleen and adrenals and increased cellularity in bone marrow (sternum and femur). Leukocytes parameter changes were reversible at the end of the recovery period. Test item related decrease in total protein/albumin/globulin, triglycerides, cholesterol and AHDL were noted in both males and females at 1000 mg/kg/day. All these changes were completely reversible except for the reduced protein profile in females at the end of the recovery period. All the changes in leukocytes parameters and protein profile were secondary to the local inflammatory condition (mucosal necrosis) noted in the non-glandular stomach and these systemic or local changes were considered non-adverse. Increased ALT levels were noticed in both sex at ≥ 300 mg/kg/day without any correlating microscopic lesions in the liver, which was considered as a non-adverse effect of the test item.

• Thyroid Hormone Profile: Thyroid hormone profile (TSH, T4 and T3) was not affected in both sexes across the treated groups when compared to the concurrent vehicle control group.

• Terminal Fasting Body Weights and Organ Weights: The decreased terminal fasting body weight was noticed in males (18%) at 1000 mg/kg/day and was reversible. At 1000 mg/kg/day, increased adrenal weights (microscopically, associated with increased extramedullary hematopoiesis); decreased thymus weights (grossly, small and microscopically, associated with decreased cellularity-cortex/medulla in few males and females) and decreased spleen (microscopically, decreased cellularity-marginal zone of white pulp in two males) and prostate weights (microscopically, atrophy with decreased secretion in a male) were noticed. Reductions in weight of spleen, thymus, heart and prostate and increased brain weights in males at 1000 mg/kg/day were considered secondary effects due to either stress or decreased feed intake and/or reduced terminal body weight. The reduced thymus weight was also noted in males at 300 mg/kg/day without any histological correlation. These organ weight changes were not considered as adverse.

• Gross pathology: Grossly, thickening of non-glandular mucosa (stomach) at 1000 mg/kg/day males and females was histologically accompanied by epithelial hyperplasia/ mucosal necrosis. The range of microscopic changes were suggestive of forestomach gastritis specifically observed in rodents and was attributed to possible irritant potential of the test item.

• Histopathology: Microscopically, degeneration of seminiferous tubules of testes (grossly- bilateral flabby testes in a male ) and atrophy of accessory sex glands- prostate, seminal vesicles and coagulating gland (grosslyprostate-small in a male) in males at 1000 mg/kg/day were noted. Epididymal cell debris were considered consequent to degenerative changes noted in testes. These changes were attributed to the significant body weight reduction observed in these animals and considered test item related secondary effects. Microscopically, increased macrophages in mesenteric lymph nodes (in cortex and paracortex region); increased extramedullary hematopoiesis in spleen (in red pulp)/adrenals (inner cortical region-zona reticularis) and increased cellularity in bone marrow of sternum/femur/marrow smear in both sex were noted at ≥ 100 mg/kg/day. The increased macrophages/extramedullary hematopoiesis/cellularity were also observed in recovery rats at the 28 day recovery period. All the above changes in pathology parameters were considered either as local response (stomach lesions) or secondary changes associated with the decreased body weight /stomach lesions and thus not considered as adverse.

No Observed Adverse Effect Level (NOAEL): As there were no biologically relevant adverse changes observed up to the highest dose, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item Reaction product of fatty acids, C18 alkyl with amines, polyethylenepoly-tetraethylenepentamine fraction is considered to 1000 mg/kg bw/day under the test conditions and doses employed.