Hexanamide, N-(2-ethylhexyl)-3,5,5-trimethyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-08-20 to 2014-08-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-ethylhexyl)isononan-1-amide

EC Number:

298-613-3

EC Name:

N-(2-ethylhexyl)isononan-1-amide

Cas Number:

93820-33-8

Molecular formula:

C17H35NO

IUPAC Name:

N-(2-Ethylhexyl)isononan-1-amide

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System

Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 7-8 weeks old, females: 7-8 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 150 - 176 g (mean: 163.70 g, ± 20% = 130.96 – 196.44 g)
females: 122 - 147 g (mean: 133.37 g, ± 20% = 106.69 – 160.04 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the
German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22+/- 3 °C
- Relative humidity: 55+/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0801)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 060613)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item formulation and vehicle were administered at a single dose to the animals by oral gavage.
The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose)
and 1 control group (C). The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days.

Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight

The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the
same volume as used for the high dose group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For determination of the concentration of test item in dosing formulations, samples of at least 25 mL were retained from all groups once weekly
during the treatment period and stored between -15 and -35 °C. In total 16 samples.
Stability of the dosing formulations was tested once at the beginning of the treatment period. From the low and high dose group samples of
dosing formulations were frozen at 0 hours and 6 hours after the preparation and stored at -15 and -35 °C. In total 4 samples.
In the 1st and 4th week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly
prepared high and low dose formulations and stored between -15 and -35 °C. In total 12 samples.
Each sample was retained twice (sample A, sample B, each of at least 25 mL).
At the end of the treatment period all A samples of dosing formulations were analysed at BSL BIOSERVICE Scientific Laboratories GmbH in
accordance with GLP under the reference number 133211.
The B samples were retained at BSL until the analysis had been performed, and will be discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days.

Frequency of treatment:

The animals were treated once daily.

Doses / concentrations

Remarks:

Doses / Concentrations:
100 mg/kg bw, 300 mg/kg bw and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5 male and 5 female animals per dose group; 5 male and 5 female animals for recovery gropus (control plus hihg dose); in total 60 animals (30 males and 30 females) were included in the study.

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random based on body weight
- Section schedule rationale : alle male animals together and all female animals together

Examinations

Observations and examinations performed and frequency:

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during
the treatment and recovery period. Food consumption was measured weekly during the treatment and recovery period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 28 days.
The recovery animals were observed for an additional period of 14 days following the last administration.
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated
effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except
on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia,
vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made
outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the
treatment period as well as at the end of the recovery period in the recovery animals.

Functional Observations
Once before the first exposure and once in the fourth week of exposure as well as in the last week of the recovery period multiple detailed
behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted
in all animals.

Haematology
Haematological parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
On the scheduled day of female necropsy, the instrument (ADVIA®120 (Siemens)) was not functioning and had to be repaired.
Blood samples collected at the terminal from female animals of main group were stored at refrigerator condition for 7 days and then measured.
All data were compared with historical control data range.
Haematological parameters (haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV),
mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT),
white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso) and large unstained cells)
were examined.

Blood Coagulation
Coagulation parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
Coagulation parameters (prothrombin time (PT), activated partial thromboplastin time (aPTT)) were examined.

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
Parameters of clinical biochemistry (alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP),
creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc),
sodium (Na), potassium (K)) were examined.

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.
The following parameters (specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood,
leukocytes) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL). Additionally, urine colour/ appearance were recorded.

Sacrifice and pathology:

Pathology
One day after the last administration (study day 29) all animals of the treatment period and 2 weeks after the last administration all animals
of the recovery period (study day 43) were sacrificed using anesthesia (ketamine, medistar Arzneimittel, Lot No: 00213, expiry date: 28.02.2015
and xylazin, Serumwerk, Lot No. 00513, expiry date: 31.05.2015) and subjected to a detailed gross necropsy which included careful examination
of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight
The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/ parathyroid glands, testes, spleen, epididymides,
brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries,heart) of all sacrificed animals was recorded as soon as possible.
Paired organs were weighed together.

The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye, uterus with cervix (females),
liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles with
coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder, thymus, lymphnodes
(mesentric and axillary), thyroid glands, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, sternum with bone marrow,
lung and trachea, pituitary gland, mammary glands, oesophagus, skin) from all animals were preserved in 10% neutral buffered formalin
except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were
transferred to 70% ethanol.

Histopathology
The afore-listed organs (see above) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin
staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period.
Because of possible treatment-related findings noted in the high dose group, liver, thyroid glands from all animals and kidneys from all
males of the low and medium dose groups, respectively as well as liver and thyroid glands from all recovery animals and kidneys from all
recovery males were examined to establish a no-effect level and the reversibility.
Any gross lesion macroscopically identified was examined microscopically in all animals.
Evaluation of sperm staging (detailed qualitative examination of testes) was carried out in all males of Control and High dose.
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services,
Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified
contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator of the test site provided the histopathology results to the study director by e-mail to be integrated in the report.
The principal investigator sent a pathology phase report to the study director.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical
biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of
the main groups using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period
were performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software or IDBS E-WorkBook software
(p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

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):

no adverse effects

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

considered male rat specific (alpha-2u globulin mediated effects)

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality
No mortality occurred in the control or any of the dose groups during the treatment period of this study.

Clinical Observations
In males and females, there were no clinical signs of toxicological relevance observed during the treatment period.
There were transient incidences of respiratory sounds, piloerection, nasal discharge, eschar and diarrhea in males or females of control
and/ or dose group animals including recovery groups. There were also cases of moving the bedding and salivation before and/or immediately
post dose administration in most males and females of the dose groups (100, 300 and 1000 mg/ kg body weight/ day), which was assumed to
be due to discomfort and not considered to be an adverse effect.
In addition, there were also isolated cases of local alopecia in a single male and a single female of the recovery group treated at 1000 mg/ kg body
weight, which was considered to be incidental. During the weekly detailed clinical observation, no significant changes or differences
between the groups were found. There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment
period in both males and females. There were no biologically relevant differences in the body temperature between the groups.

Body Weight Development
In males and females, there were no adverse changes of toxicological relevance noted for the body weight and body weight gain during the
treatment and recovery period. There were no statistically significant changes noted.
In males and/ or females there was slight to moderate increase/ decrease in weight gain after 3rd week of treatment at the dose levels
100, 300 and/ or 1000 mg/ kg body weight/ day when compared to control. In the recovery HD group males and/ or females there was slight
to marked increase and slight to marked decrease in weight gain after the initial weeks (1st and 2nd) of treatment and during the recovery period,
respectively when compared to concurrent control. The increases and/ or decreases in weight gain were not statistically significant and in
addition did not correspond to the food intake. Therefore, the increase or decrease in weight gain was not considered to be an adverse effect
of treatment.

Food Consumption
In males and females, there were no changes of toxicological relevance noted for food consumption during the treatment and the
recovery period.In females there was slight decrease in food consumption noted after the 2nd week of treatment at
100 mg/ kg body weight/ day when compared to control. There was no statistically significant decrease and/ or dose related response noted.

Haematology and Blood Coagulation
In males and females, there were no adverse changes of toxicological relevance noted for haematology and blood coagulation parameters
at the end of the treatment and recovery period. In males there was statistically significant decrease in mean reticulocyte count at 1000 mg/ kg
body weight/ day at the end of treatment when compared to control. This change in reticulocyte count was not accompanied by change in red
blood cell and white blood cells counts which were comparable to the corresponding concurrent control group. In addition the mean and
individual values of reticulocyte count were within the historical control data range.
There were slight increases in the mean values of haemoglobin (Hb), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin
concentration (MCHC) in males at 1000 mg/ kg body weight/ day at the end of the recovery period when compared to concurrent control,
but the increases were not statistically significant and in addition all mean and individual values were within the historical control data range.
In females there were statistically significant increases in neutrophil and decrease in lymphocyte count at 1000 mg/ kg body weight at the
end of treatment when compared to control. There was also a slight but not statistically significant increase in eosinophil count at 1000 mg/ kg
body weight/ day. The neutrophil, lymphocyte and eosinophil counts at 1000 mg/ kg body weight/ day at the end of the recovery period were
comparable to the concurrent control group. The mean and individual values of neutrophil, lymphocyte and eosinophil values were within
the historical control data range. The statistical significance of neutrophil and lymphocyte count at high dose group had no biological relevance.
There was also statistically significant increase in mean prothrombin time in females at 1000 mg/ kg body weight/ day at the end of recovery
period when compared to control. The mean prothrombin time value was close to concurrent control value and in addition the mean and
individual prothrombin time values were within the historical control data range. Hence, the statistical significance of prothrombin
time value had no biological relevance.

Clinical Biochemistry
In males and females, there were no adverse changes of toxicological relevance noted for clinical biochemistry parameters at the end of
treatment and recovery period. In males, at the end of treatment there were statistically significant decreases in alanine aminotransferase,
alkaline, phosphatase and glucose at 1000 mg/ kg body weight / day and in total bile acids at 100, 300 and 1000 mg/ kg body weight/ day;
statistically significant decrease in urea at 300 and 1000 mg/ kg body weight/ day when compared to control.
At the end of recovery, there was also statistically significant decrease in glucose at 1000 mg/ kg body weight/ day. The mean and most
individual values of alanine aminotransferase, alkaline phosphatase, glucose and total bile acids were within the historical control data range.
In addition the slight decrease in glucose and total bile acids in animals could be an indication of prolonged fasting before necropsy.
There was also statistically significant increase in mean sodium at 1000 mg/ kg body weight/ day, but the mean value was close to the
concurrent control value and in addition the mean and individual values were within the historical control range.
In females, at the end of treatment there was statistically significant increase in alanine aminotransferase and cholesterol values at
1000 mg/ kg body weight/ day and statistically significant increase in albumin and sodium values at 300 and 1000 mg/ kg body weight/ day
when compared to control. The increase in alanine aminotransferase and albumin values did not show a dose response relation.
The changes in mean sodium values were close to the concurrent control and the recovery control values. In addition the mean and individual
values were within the historical control range. Hence, the statistical significance had no biological relevance.
The serum cholesterol values in female dose groups showed dose related response. All mean and individual cholesterol values of dose
groups including the control were above the historical control data range. The increase in cholesterol in female dose groups reversed at the
end of recovery and was comparable to control. The increase in cholesterol was correlated to the adaptive changes in liver
(centrilobular hepatocellular hypertrophy), which was not considered to be an adverse effect of treatment.
There were also decreases, not statistically significant, in alkaline phosphatase and total bile acids at 300 and 1000 mg/ kg body weight/ day.
The mean and individual values were within the historical control range and therefore the decrease in alkaline phosphatase and total
bile acids had no biological relevance.

Urinalysis
There were no adverse treatment related changes of urine parameters for males and females at the end of the treatment or the recovery period.
However, there were slightly elevated levels of erythrocytes in males at 1000 mg/ kg body weight/ day at the end of treatment and recovery period. The findings noted in the males were supported by histopathological kidney changes. The microscopic changes of kidneys were deemed to be
unlikely to occur in humans. Therefore, the slightly elevated levels of erythrocytes in male at 1000 mg/ kg body weight/ day were not
considered to be an adverse effect.

Pathology
There were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded were considered to be within
the range of normal background alterations.

Organ Weight
In males and females, there were no adverse changes of toxicological relevance noted for absolute and relative organ weights.
At the end of treatment there were statistically significant increases noted for absolute and/ or relative (to brain and/ or body weight) liver,
kidneys and thyroid weights of males and/ or females at 300 and/ or 1000 mg/ kg body weight/ day when compared to control. There was also
statistically significant decrease in absolute prostate (including seminal vesicle+ coagulating glands) weight in male at
1000 mg/ kg body weight/ day.
At the end of recovery, in males there were statistically significant increases in relative (to body weight) thyroid weight and absolute and relative
(to brain and body weight) thymus weights when compared to concurrent control. There was also statistically significant increase in absolute heart
weight and relative (to brain and body weight) kidney weights in female at the end of recovery.
There were also some variations (increase or decrease) from corresponding concurrent control group noted for spleen, thymus, uterus with
cervix and adrenal weights (absolute and/ or relative) in treated and/ or recovery groups of male or female animals, but the changes were
not statistically significant.
The organ weight changes of liver, kidneys and thyroid in male or female animals at 300 and/ or 1000 mg/ kg body weight/ day were
supported by microscopic changes in liver, thyroid and kidneys noted in both sexes except kidneys where the structural changes were noted
only in male dose groups. Based on the microscopic findings, the changes in organ weight were not considered to be an adverse effect of treatment.
The absolute and relative weight changes in rest organs in the absence of any supporting histopathological changes were not considered to
be toxicologically relevant.


Histopathology
The histopathological evaluation of organs/ tissues revealed centrilobular hepatocellular hypertrophy of liver in both sexes at 300 and
1000 mg/ kg body weight/ day, and this increased in incidence and/or severity was in a dose-dependent manner. There were no further
indicators of liver injury, and therefore this finding was considered to be of adaptive character and not to be adverse under the condition of
this study.
In this study thyroid follicular cell hypertrophy was recorded at minimal severity in both sexes at 1000 mg/ kg body weight. This change is
often elicited by the increase of thyroid hormones (T3/T4) metabolism in the liver, and a possible relationship between the liver enzyme
induction and these histomorphologic changes of liver and thyroid glands was considered. Accordingly, thyroid follicular cell hypertrophy
was considered to represent a secondary effect, and hence, not to be adverse.
Increased incidence and/or severity of hyaline droplets in proximal tubules were recorded in males of all test item treatment groups with a
dose-dependent manner. Since hyaline droplets were not observed in any of the high-dose females, this was considered to contain a male
rat-specific protein like alpha-2u globulin. In addition, there were no further indicators of renal injury. Therefore, this change was considered
not to be adverse under the condition of this study, and deemed to be unlikely to occur in humans. 
Hepatocellular hypertrophy and thyroid follicular hypertrophy was no longer present in recovery animals. Hyaline droplets regressed after the
treatment-free recovery period.
As a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology
including spermatogenesis and interstitial cell structure.


Dose Formulation Analysis
for all dose groups. The mean recoveries observed at dose levels 100, 300 and 1000 mg/kg body weight/ day were 100.9%, 97.9% and 96.6% of
the nominal concentration, respectively.
Stability of formulation samples was investigated in study week 1 for dose levels 100 and 1000 mg/ kg body weight/ day. After 6 hours storage
at room temperature recovery compared to starting value was between 98.4% and 104.0%.
Homogeneity of formulation samples was determined in study week 1 and 4 for dose levels 100 and 1000 mg/ kg body weight/ day. The mean
recovery observed for dose level 100 mg/ kg body weight/ day was between 107.6% and 89.0% of the nominal value, and between 112.5 and 88.9%
of the nominal value for dose level 1000 mg/ kg body weight/ day. The coefficients of variation of the different sampling locations (top, middle,
bottom) were between 2.8% and 3.7% at dose level 100 mg/ kg body weight/ day, and between 5.1% and 6.2% at dose level 1000 mg/ kg body
weight/ day.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the NOAEL (no-observed-adverse-effect- level) was considered to be 1000 mg/ kg body weight/ day for both males and females.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of this 28-Day Repeated Dose Oral Toxicity study with “N-(2-Ethylhexyl)isononan-1-amide” in male and female Wistar rats with dose
levels of 100, 300, and 1000 mg/kg body weight/ day, followed by a recovery period of 14 days, the following conclusions can be made:
There were slight alteration in body weight in males and females at or above 300 mg/kg body weight/ day, decrease in reticulocyte count in males,
increase and decrease in neutrophil and lymphocytes, respectively in females at 1000 mg/ kg body weight/ day, increase in alanine aminotransferase, cholesterol, albumin and sodium values in females at or above 300 mg/kg body weight/ day, elevated erythrocyte count in urine of males at
1000 mg/ kg body weight/ day, increase in organ weight in males and/ or females at or above 300 mg/ kg body weight/ day, centrilobular
hepatocellular hypertrophy of liver, thyroid follicular cell hypertrophy at or above 300 mg/ kg body weight/ day
and increased incidence and/or severity of hyaline droplets in proximal tubules in males of all dose groups.
All above changes were not considered to be an adverse effect of the test item treatment and/or without toxicological relevance for humans.
Hence, the NOAEL (no-observed-adverse-effect- level) was considered to be 1000 mg/ kg body weight/ day for both males and females.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of “N-(2-Ethylhexyl)isononan-1-amide”via oral administration to rats over a period of 28 days.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study.

The 4 groups comprised of 5 male and 5 femaleWistarrats.

During the period of administration, the animals were observed precisely each day for signs of toxicity. All animals were sacrificed and

observed macroscopically. To detect possible delayed occurrence or persistence of, or recovery from toxic effects, animals in the recovery

group (5 animals/ sex in control and HD groups) were observed for a period of 14 days following the last administration.

The following doses were evaluated:

Control:                           0         mg/kg body weight/ day

Low Dose:                    100     mg/kg body weight/ day

Medium Dose:              300    mg/kg body weight/ day

High Dose:                   1000  mg/kg body weight/ day

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in corn oil and administered daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements. The animals were treated at the dose volume of 4 mL/ kg body weight.

Body weight and food consumption were measured weekly.

At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. Blood samples were collected from the abdominal aorta for the evaluation of hematology, blood coagulation and clinical biochemistry. The urine sample was collected from the urinary bladder for the urinalysis.

A full histopathological evaluation of the tissues was performed on high dose and control animals.Because of the possible treatment-related findings noted in the high dose group, liver, thyroid glands from all animals and kidneys from all males of the low and intermediate groups (100 and 300 mg/ kg body weight/ day, respectively) as well as liver and thyroid glands from all recovery animals and kidneys from all recovery males were examined to establish a no-effect level and the reversibility.Gross lesion macroscopically identified was examined microscopically in all animals.

Summary Results

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

In males and females, there were no clinical signs of toxicological relevance observed during the treatment period. There were few incidences of moving the bedding and salivation before and/or immediately post dose administration in most animals (males and females) of the dose groups (100, 300 and 1000 mg/ kg body weight/ day), which was assumed be due to discomfort and not considered to be an adverse effect of the treatment.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

There were no ophthalmoscopic findings in any of the animals of this study.No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period in both males and females. There were no biologically relevant differences in the body temperature between the groups.

In males and females, there were no adverse changes of toxicological relevance noted for the body weight and body weight gain during the treatment and recovery period.

In males and females, there were no changes of toxicological relevance noted for food consumption during the treatment and the recovery period.

In males and females, there were no adverse changes of toxicological relevance noted for haematology, blood coagulation and clinical biochemistry parameters at the end of the treatment and recovery period.

There were no adverse treatment related changes noted for urine parameters noted for males and females at the end of the treatment or the recovery period.

There were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded were considered to be within the range of normal background alterations.

In males and females, there were no adverse changes of toxicological relevance noted for absolute and relative organ weights. There were increase in liver, kidneys and thyroid in male or female animals at 300 and/ or 1000 mg/ kg body weight/ day which corresponded to microscopic changes noted in liver and thyroid gland noted in both sexes and kidneys only in male dose groups.

Under the conditions of thisstudy,the test item N-(2-Ethylhexyl)isononan-1-amide produced histomorphologic changes in liver and thyroid glands of both sexes and in kidneys of males. In the liver,centrilobular hepatocellular hypertrophy was recorded in both sexes at300and1000 mg/kg body weight/ day,thyroid follicular cell hypertrophy was recorded at minimal severity in both sexes at 1000 mg/kg/day.The thyroid follicular cell hypertrophy was considered to represent a secondary alteration to enzyme induction in the liver.Increased incidence and/or severity of hyaline droplets in proximal tubules were recorded in males of all test item treatment groups with a dose-dependent manner. Since hyaline droplets were not observed in any of the high-dose females, this was considered to contain a male rat-specific protein like alpha-2u globulin. In addition, there were no further indicators of renal injury.Hepatocellular hypertrophy and thyroid follicular hypertrophy werenolonger present in recovery animals.Hyaline droplets regressed after the treatment-free recovery period.As a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

Formulation samples analysis was determined on samples collected at various intervals during the study period. For concentration analysis (in study week 1, 2, 3 and 4 for all dose groups) the mean recoveries observed in groups treated at 100, 300 and 1000 mg/kg body weight/ day were 100.9%, 97.9% and 96.6% of the nominal concentration, respectively. For stability of formulation samples (investigated in study week 1 for groups treated at 100 and 1000 mg/kg body weight/ day) after 6 hours storage at room temperature the recovery compared to starting value was between 98.4% and 104.0%. For homogeneity of formulation samples (determined in study week 1 and 4 for groups treated at 100 and 1000 mg/kg body weight/ day) the mean recovery observed fordose grouptreated at 100 mg/ kg body weight/ day was between 107.6% and 89.0% of the nominal value, and between 112.5 and 88.9% of the nominal value for dose group treated at 1000 mg/ kg body weight/ day. The coefficients of variation of the different sampling locations (top, middle and bottom) were between 2.8% and 3.7% for group treated at 100 mg/ kg body weight/ day, and between 5.1% and 6.2% for group treated at 1000 mg/ kg body weight/ day.

Conclusion

On the basis of this 28-Day Repeated Dose Oral Toxicity study with “N-(2-Ethylhexyl)isononan-1-amide” in male and femaleWistarrats with dose levels of 100, 300, and 1000 mg/kgbody weight/ day, followed by a recovery period of 14 days, the following conclusions can be made:

There were slight alteration in body weight in males and females at or above 300 mg/kg body weight/ day, decrease in reticulocyte count in males, increase and decrease in neutrophil and lymphocytes, respectively in females at 1000 mg/ kg body weight/ day, increase inalanine aminotransferase,cholesterol,albuminand sodium values in females at or above 300 mg/kg body weight/ day, elevated erythrocyte count in urine of males at 1000 mg/ kg body weight/ day, increase in organ weight in males and/ or females at or above 300 mg/ kg body weight/ day,centrilobular hepatocellular hypertrophy of liver, thyroid follicular cell hypertrophy recorded in males and females at or above 300 mg/ kg body weight/ day and increased incidence and/or severity of hyaline droplets in proximal tubules in males of all dose groups.

All above changes were not considered to be an adverse effect of the test item treatmentand/or without toxicological relevance for humans.Hence, the NOAEL (no-observed-adverse-effect- level) was considered to be 1000 mg/ kg body weight/ day for both males and females.