3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 14 May 2020 to March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH


Target : RN CAS 59219-71-5 - EC number : 261-665-2 - chemical name : 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate
Source 1 : RN CAS (previously 42131-25-9)- EC number: 701-133-3 - chemical name : Isononyl isononanoate or branched-nonyl 3,5,5 trimethylhexanoate

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
See attaced document section 13 - Rationale & justification for the Analogue read across approach used in the registration dossier of 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate
CAS 59219-71-5

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See attaced document section 13 - Rationale & justification for the Analogue read across approach used in the registration dossier of 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate
CAS 59219-71-5

3. ANALOGUE APPROACH JUSTIFICATION
See attaced document section 13 - Rationale & justification for the Analogue read across approach used in the registration dossier of 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate
CAS 59219-71-5

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH


Target : RN CAS 59219-71-5 - EC number : 261-665-2 - chemical name : 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate
Source 1 : RN CAS (previously 42131-25-9)- EC number: 701-133-3 - chemical name : Isononyl isononanoate or branched-nonyl 3,5,5 trimethylhexanoate

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
See attaced document section 13 - Rationale & justification for the Analogue read across approach used in the registration dossier of 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate
CAS 59219-71-5

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See attaced document section 13 - Rationale & justification for the Analogue read across approach used in the registration dossier of 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate
CAS 59219-71-5

3. ANALOGUE APPROACH JUSTIFICATION
See attaced document section 13 - Rationale & justification for the Analogue read across approach used in the registration dossier of 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate
CAS 59219-71-5

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 600 mg/kg bw/day (nominal)

Based on:

other: Isononyl isononanoate or branched-nonyl 3,5,5 trimethylhexanoate

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline No. 408, Repeated Dose 90-Day Oral Toxicity Study in Rodents, 25 Jun 2018

Principles of method if other than guideline:

A justification can be found under "Justification for type of information"

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

42131-25-9

EC Number:

609-993-0

Cas Number:

42131-25-9

Molecular formula:

Not relevant for UVCB

IUPAC Name:

42131-25-9

Test material form:

other: viscous liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Branched-nonyl 3, 5, 5, trimethylhexanoate. Two transparent glass flasks and two transparent plastic flasks - Batch No.: 19110632
- Purity, including information on contaminants, isomers, etc.: - Source (i.e. manufacturer or supplier) and lot/batch number of test material: 100%,
- Storage condition of test material: At room temperature and protected from light
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: According to Charles River Laboratories Evreux in-house procedures, the test item was weighed and mixed with the required quantity of vehicle (tendency to adsorb on glass: solvent was added first). The solution was mixed and stirred for at least 15 minutes
Test item dose formulations:
• on the days of treatment
Control dose formulation:
• according to the vehicle expiry date
Storage : At room temperature and protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley, Crl:CD®(SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 106 rats (53 males and 53 females) were received at Charles River Laboratories Evreux on 14 May 2020. Breeder: Charles River Laboratories Italia, Calco, Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age/Weight: on the first day of treatment, the animals were 5 to 6 weeks old. The males had a mean body weight of 230.7 g (range: 193.8 g to 282.2 g) and the females had a mean body weight of 164.1g (range: 141.8 g to 194.3 g). At the beginning of the treatment period the weight variation of animals did not exceed ± 20% of the mean weight of each sex - Fasting period before study:
- Housing: The animals were group-housed by two or three animals from the same sex and group (see Section 6.13.), in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust (Le comptoir des sciures, Meyzieu, France).
Animals were isolated where experimental procedure dictated and when an animal showed clinical signs or was due for clinical and/or laboratory investigations. This was documented in the study file.
Each cage contained at least two objects (e.g. rat hut, nylabone, wood stick) for environmental enrichment.
The cages were placed in numerical order on the racks. In Weeks 9 and 14, all the racks were moved clockwise around the room, rack by rack. In this way, for each group, identical exposure to environmental conditions was achieved.
- Diet (e.g. ad libitum): All animals had free access to SSNIFF rat/mouse pelleted maintenance diet, batch Nos. 63860736 and 57266070 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter). During periods of fasting, food but not water was removed.
- Acclimation period: the animals were acclimated to the study conditions for 13 days before the beginning of the treatment period. Three supernumerary animals/sex were allocated to the study and acclimated, in order to permit the selection and/or replacement of individuals.

DETAILS OF FOOD AND WATER QUALITY:
The batches of diet and sawdust were analyzed by the Suppliers for composition and contaminant levels.
Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 14 may 2020 To: 23 September 2020

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since it is a route of administration which is requested by the Regulatory Authorities for this type of test item.
The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, in the morning (except on the day of FOB tests, when treatment was performed in the afternoon).
The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
A constant dosage volume of 5 mL/kg/day was used.
Control animals (group 1) received the vehicle only.
The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the administration procedure.
The control and test item dose formulations were stirred just before administration and were maintained under continuous magnetic stirring throughout the administration procedure

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, in the morning (except on the day of FOB tests, when treatment was performed in the afternoon).
The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
A constant dosage volume of 5 mL/kg/day was used.
Control animals (group 1) received the vehicle only.
The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the administration procedure.
The control and test item dose formulations were stirred just before administration and were maintained under continuous magnetic stirring throughout the administration procedure
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0, 20, 60, 120 mg/mL that corresponds to 0, 100, 300 and 600 mg/kg/day
- Amount of vehicle (if gavage): A constant dosage volume of 5 mL/kg/day was used.
- Lot/batch no. (if required): MKCK6411: 14 and 29 May 2021, 18 Aug 2021, MKCD1021: 04 Sep 2020, MKCH1635: 13 Dec 2020

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The control and test item dose formulations were stirred just before administration and were maintained under continuous magnetic stirring throughout the administration procedure.
• the determination of test item concentrations in dose formulations was performed as planned, once at the beginning and end of the treatment period and once during the course of the study, i.e. total of 3 occasions instead of 4 as mistakenly stated in amendment No. 1,
• at the beginning of the treatment period, determination of test item concentration in group 2 dose formulation was performed on three samples instead of one to secure the test due to a slight delay in the analytical method validation for the low-dose formulation, delay which was caught up concomitantly.

Duration of treatment / exposure:

The dose formulations were administered daily for 13 weeks.
At the end of the treatment period, animals in each group were euthanized, except for the first five animals of both sex in groups 1 and 4 (recovery animals), which were kept for a 4 week treatment-free period.
Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, in the morning (except on the day of FOB tests, when treatment was performed in the afternoon).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

600 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Number and sex of animals
• Dose level (0 mg/kg/day)
15 males /15 females
Animal number 1001-1015 / 1501-1515

• Dose level (100 mg/kg/day)
10 males/10 females
Animal number 2001-2010 / 2501-2510

• Dose level (300 mg/kg/day)
10 males/ 10 females
Animal number 3001-3010/3501-3510

• Dose level (600 mg/kg/day)
15 males/15 females
Animal number 4001-4015/ 4501-4515 600

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels have been selected in agreement with the Sponsor, based on the results of a previous study (Study No. 48231 TSR (Palmieri, 2020)) where dose levels of 100, 300 and 600 mg/kg/day were administered for four weeks. Following the administration during four weeks, no clinical effects were observed and microscopic findings were limited to non-adverse microscopic hepatocellular hypertrophy and/or vacuolation at all doses.
Based on these results, the same dose levels of 100, 300 and 600 mg/kg/day were selected for the present study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed for the recording of clinical signs:
• at least once a day during the treatment period at approximately the same time of the day (between 1 and 3 hours post-dosing),
• at least once a day at approximately the same time of the day during the treatment-free period
- Cage side observations checked in table.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
• once before the beginning of the treatment period,
• at least once a week during the treatment period,
• once a week during the treatment-free period
Each animal was checked for mortality and morbidity at least once a day during the acclimation period, at least twice a day during the treatment period, then at least twice a day during the treatment-free period.
The following parameters were assessed and graded:
• in the cage: "touch escape",
• in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
• in the standard arena (two-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.


BODY WEIGHT: Yes
- Time schedule for examinations:
• at least once a week before the beginning of the treatment period,
• on the first day of treatment,
• at least once a week until the end of the study.


FOOD EFFICIENCY:
The quantity of food consumed by the animals in each cage was recorded at least once a week from the first day of treatment until the end of the study.
Food consumption was calculated per animal and per day. When one of the animals in the same cage died, the number of days for which that animal had been present in the cage was taken into consideration for the calculation of food consumption.
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were performed:
• on all animals, before the beginning of the treatment period,
• on control and high-dose animals on one occasion during Week 12.
As no changes were reported at the end of the treatment period, these examinations were not carried out in low- and mid-dose groups at the end of the treatment period or at the end of the treatment-free period.
The pupils of the animals were dilated with tropicamide (Mydriaticum®, Laboratoires Théa, Clermont-Ferrand, France). After assessment of the corneal reflex (at instillation of the tropicamide), the appendages, optic media and fundus were examined by indirect ophthalmoscopy.
When necessary, a slit-lamp biomicroscope was used to investigate abnormalities of the anterior segment and the lens.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals:
- Parameters checked in table.
The following parameters were determined for all surviving animals (excluding recovery animals) during Week 13.
In view of the findings observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.
• Blood was collected into BD Microtainer® (K2EDTA) tubes and analyzed using the ADVIA 120 Hematology analyzer: erythrocytes (RBC)
Mean Cell Volume (MCV)
packed cell volume (HCT)
hemoglobin (HGB)
Mean Cell Hemoglobin Concentration (MCHC)
Mean Cell Hemoglobin (MCH)
thrombocytes (PLT)
leucocytes (WBC)
Differential white cell count with cell morphology
. neutrophils (NEUT)
. eosinophils (EOS)
. basophils (BASO)
. lymphocytes (LYMP)
. large unstained cells (LUC)
. monocytes (MONO)
reticulocytes (%RETI)
reticulocytes (RTC)
A blood smear for possible determination of the differential white cell count (with cell morphology) was prepared for each animal and stained with May Grünwald Giemsa.
As all the blood samples were successfully analyzed by the ADVIA 120, the blood smears were archived without further investigation.
A blood smear (stained with cresyl blue) for possible determination of the reticulocyte count was prepared for each animal.
As all the blood samples were successfully analyzed by the ADVIA 120 the blood smears were archived without further investigation.
Prior to blood sampling and during urine collection, the animals were deprived of food for an overnight period of at least 14 hours but not exceeding 18 hours.
Blood samples were collected from the jugular vein into tubes containing the appropriate anticoagulant.
For urine collection the animals were placed in individual metabolism cages for an overnight period of at least 14 hours. The urine was collected onto thymol crystals.
Bone Marrow
Two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals euthanized on completion of the treatment or treatment-free period and stained with May Grünwald Giemsa.
As no hematological abnormalities requiring a bone marrow differential cell count were observed, the smears were archived without further investigations.
Coagulation
The following parameters were determined for all surviving animals (excluding recovery animals) during Week 13.
In absence of test item-related changes at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.
Blood was collected into sodium citrate tubes (plasma was separated within 90 minutes of blood collection -and analyzed using ACL TOP 550 CTS blood coagulation analyzer: Prothrombin Time (PT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- Parameters checked in table were examined.
The following parameters were determined for all surviving animals (excluding recovery animals) during Week 13.
In view of the findings observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.
• Blood was collected into lithium heparin tubes (plasma was separated within 90 minutes of blood collection) and analyzed using the ADVIA 1800 blood biochemistry analyzer:
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (CA)
Inorganic phosphorus (PHOS)
Glucose (GLUC)
Urea (UREA)
Creatinine (CREA)
Total bilirubin (BILT)
Total cholesterol (CHOL)
HDL cholesterol (HDL)
LDL cholesterol (LDL)
Triglycerides (TRIG)
Bile acids (AcBIL)
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Total proteins (PROT)
Albumin (ALB)
Albumin/Globulin ratio (A/G)

PLASMA/SERUM HORMONES/LIPIDS: Yes
Thyroid Hormones
An additional blood sample was collected, at a comparable time in the first half of the morning (between 7.5 and 10 a.m ) from all surviving animals (excluding recovery animals) at the end of the treatment period and from all surviving animals at the end of the treatment-free period into tubes containing K3EDTA as anticoagulant.
The blood was centrifuged within 2 hours after sampling (approximately 3000g for 10 minutes at +4°C). The plasma was transferred into 2 separate tubes (at least 125 µL in the first tube and the remaining plasma in the second tube) and frozen at -80°C pending analysis.
The levels of the thyroid hormones (T3 and T4) were determined by LC-MS/MS method and Thyroid Stimulating Hormone (TSH) was determined by Luminex MAP® technology for all surviving animals euthanized at the end of the treatment or treatment-free period.


URINALYSIS: Yes
- Time schedule for collection of urine: The following parameters were determined for all surviving animals (excluding recovery animals) during Week 13.
In view of the findings observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.
- Animals fasted: Yes
- Parameters checked in table were examined.
volume (UVol)
pH (UpH)
Specific gravity (UGrav)
Proteins (UProt)
Glucose (UGluc)
Ketones (UCeto)
Bilirubin (UBili)
Nitrites (UNitr)
Blood (hemoglobin) (UBlood)
Cytology of sediment
. leucocytes (WBC-U)
. erythrocytes (RBC-U)
. cylinders (CY-U)
. magnesium ammonium phosphate crystals (CR-PA)
. calcium phosphate crystals (CR-PC)
. calcium oxalate crystals (CR-OC)
. epithelial cells (CE-U)
Appearance (UCla)
Color (UColor)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested:
• touch response,
• forelimb grip strength,
• pupillary reflex,
• visual stimulus,
• auditory startle reflex,
• tail pinch response,
• righting reflex,
• landing foot splay,
• at the end of the observation period: rectal temperature
For each animal, motor activity was measured by automated infra-red sensor equipment over a 60-minute period

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Following the observation of dorso-cervical scabs, an antiseptic solution (Cothivet®) was locally applied, as follows:
• male 1007 and female 1512 (group 1): once a day for 8 days starting on Day 15,
• male 1014 (group 1): once a day for 13 days starting on Day 17,
• male 1011 (group 1): twice a day for 10 days starting on Day 35.
On Day 64, the wound on the right hindlimb of female 2507 (group 2) was disinfected with Cothivet®.
On Day 88, following the observation of a wound on the tail of female 3508 (group 3), an antiseptic solution (Vétédine®) was applied once a day for 5 days.

Monitoring of Estrous Cycle
The estrous cycle stage was determined from daily vaginal smears during three weeks of the treatment period.
In absence of test item-related changes during the treatment period, these examinations were not carried out at the end of the treatment-free period.
Only individual estrous cycle data are presented in the study report. No statistical analyses were performed on those data

Sperm Parameters
At the end of the treatment period, sperm sampling was performed during euthanasia, after intraperitoneal injection of sodium pentobarbital.
In absence of test item-related changes at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.
Epididimal Sperm
After the intraperitoneal injection of sodium pentobarbital and epididymis weighing, sperm from the cauda of the left epididymis was sampled for motility and morphology investigations
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at -20°C pending further investigation.

Epididymal Sperm Motility
The sperm was evaluated on a slide, after appropriate dilution if needed. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification for all groups. Results are expressed as the proportion of motile and non-motile spermatozoa.

Epididymal Sperm Morphology
For control and high-dose groups, the morphology was determined from a sperm smear, after eosin staining and counting of 200 spermatozoa per slide. Results are expressed as the proportion of spermatozoa in each of the following categories:
• normal,
• normally shaped head separated from flagellum,
• abnormal head separated from flagellum,
• abnormal head with normal flagellum,
• abnormal head with abnormal flagellum,
• normally shaped head with abnormal flagellum.

Epididymal Sperm Count
For control and high-dose groups, after thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results are expressed as the number of spermatozoa per cauda and per gram of cauda.

Testicular Sperm
For control and high-dose groups, the left testis was sampled and frozen at -20°C for further sperm count investigation. After thawing, the left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber.
Results are expressed as a number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10; Blazak et al., 1993).

Sacrifice and pathology:

Euthanasia
On completion of the treatment or treatment-free period, after at least 14 hours fasting, all surviving animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination.
Sperm sampling was performed during euthanasia at the end of the treatment period, after intraperitoneal injection of sodium pentobarbital.

Organ Weight
The body weight of each animal was recorded before euthanasia at the end of the treatment or treatment-free period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.
Macroscopic Post-Mortem Examination
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

Preservation of Tissues
For all study animals, the tissues specified in the Tissue Procedures Table were preserved in 10% buffered formalin (except for the eyes and optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).
Tissues intended for immunohistochemistry (kidneys) were kept for no longer than 96 hours in formalin (all males).
Two bone marrow smears for potential determination of the bone marrow differential cell count were prepared from the femur of each animal euthanized on completion of the treatment or treatment-free period.

Preparation of Histological Slides
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert, et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS).
One additional kidney slide of all males from the control and high-dose levels groups euthanized at the end of the treatment period or treatment-free period was immunostained with an antibody for Alpha2u-globulin.

Microscopic Examination
A microscopic examination was performed on:
• all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period and for female 4515 dead during blood sampling,
• all macroscopic lesions, thymus, spleen, stomach (females only), thyroids (males only), kidneys and liver from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period,
• thymus, spleen, stomach (females only), thyroids (males only), liver and kidneys for recovery animals.
In addition, a detailed examination of the testes was performed for control and high-dose males (groups 1 and 4), using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin-PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
In addition, according to kidney histopathology, immunostained kidneys from all males from the control and high-dose level groups euthanized at the end of the treatment and treatment-free period were examined .
This was performed by the Principal Investigator, Loïc Longeart, under the responsibility of Charles River Laboratories Evreux.

Preservation of tissues :
Adrenals
Aorta
Brain (including medulla/pons cerebella and cerebral cortex)
Cecum
Colon
Duodenum
Epididymides: right (a)
. left (a)
Esophagus
Eyes with Harderian glands
Femoral bone with articulation
Gut-Associated Lymphoid Tissue (GALT)
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs with bronchi
Lymph nodes (mesenteric, mandibular)
Mammary gland area
Optic nerves
Ovaries (with oviducts)
Pancreas
Pituitary gland
Prostate (dorsolateral and ventral) (b)
Rectum
Salivary glands (sublingual and submandibular)
Sciatic nerve
Seminal vesicles (including coagulating glands) (b)
Skeletal muscle
Skin
Spinal cord (cervical, thoracic and lumbar)
Spleen
Sternum with bone marrow
Stomach with forestomach
Testes:. right (a) / left (a)
Thymus
Thyroids with parathyroids
Tongue
Trachea
Urinary bladder
Uterus (horns and cervix)
Vagina

Organs weights
Adrenals
Brain (including medulla/pons cerebella and cerebral cortex)
Epididymides: right (a)/ left (a)
Heart
Kidneys
Liver
Ovaries (with oviducts)
Pituitary gland
Prostate (dorsolateral and ventral) (b)
Skeletal muscle
Spleen
Testes:. right (a) / left (a)
Thymus
Thyroids with parathyroids
Uterus (horns and cervix)

Microscopic examination
Adrenals
Aorta
Brain (including medulla/pons cerebella and cerebral cortex)
Cecum
Colon
Duodenum
Epididymides: right (a)/ left (a)
Esophagus
Femoral bone with articulation
Gut-Associated Lymphoid Tissue (GALT)
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs with bronchi
Lymph nodes (mesenteric, mandibular)
Mammary gland area
Ovaries (with oviducts)
Pancreas
Pituitary gland
Prostate (dorsolateral and ventral) (b)
Rectum
Salivary glands (sublingual and submandibular)
Sciatic nerve
Seminal vesicles (including coagulating glands) (b)
Skeletal muscle
Skin
Spinal cord (cervical, thoracic and lumbar)
Spleen
Sternum with bone marrow
Stomach with forestomach
Testes:. right (a) / left (a)
Thymus
Thyroids with parathyroids
Trachea
Urinary bladder
Uterus (horns and cervix)
Vagina
(a): when sperm parameters were performed. When sperm parameters were not performed, left part was preserved. In both cases, right and left organs were weighed together; (b): first the prostate was weighed together with seminal vesicles/coagulation glands, then the prostate was weighed separately after dissection.

Other examinations:

see examination section

Statistics:

Provantis software was used to perform the statistical analysis of body weight, food consumption, sperm parameters, motor activity, hematology, coagulation, blood biochemistry, urinalysis and thyroid hormone.
PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Single to repeated occurrences of minimal to severe hypersalivation were observed during and/or at the end of the treatment period in a few males at ≥ 300 mg/kg/day (1/10 males at 300 mg/kg/day, 4/15 males at 600 mg/kg/day) and females at 600 mg/kg/day (6/14 surviving females). As not observed in controls or at lower doses, this finding was attributed to the test item. Hypersalivation, commonly observed with no impact on the general condition of the animals when a test item is administered by gavage, was considered of no toxicological importance. This was no longer observed during the treatment-free period and was therefore considered to be reversible.
All other clinical signs observed during the study (i.e. reflux at dosing, chromodacryorrhea, chromorhynorrhea, soiled coat, alopecia, scabs/wounds, thin or erected fur, bent tail, pallor, thin appearance, hunched posture or decreased activity) were considered incidental as they were occasional and/or observed both in control and test item-treated animals at similar frequency, incidence and severity and/or can commonly be encountered in group-housed laboratory rats dosed by gavage.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test item-related deaths.
Female 4515 (600 mg/kg/day) died on Day 87, during blood sampling for laboratory investigations at the end of the treatment period. Except for hypersalivation, observed from Day 78, there were no clinical signs before death. The most important macroscopical observation was a subcutaneous red and gelatinous ventral thoracic area measuring about 10 x 4 cm. This correlated with marked hemorrhage. The cause of demise was considered to be traumatic and unrelated to the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on body weights or body weight gains.
Any differences between control and test item-treated groups, including those that were statistically significant, were transient, observed sporadically with no dose relationship and/or were consistent with normal biological variations, and were therefore not considered to be test item-related

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

When compared to controls, occurrences of slightly higher mean food consumption were often reported during the treatment period in males and females at 600 mg/kg/day, persisting in males during the treatment-free period. While a relationship to the test item-treatment could not be excluded, these differences from controls were of slight magnitude and had no impact on the final body weights, and were thus considered to be of no toxicological importance.
Any other differences between control and test item-treated groups, including those resulting in statistical differences, were transient and/or consistent with normal biological variation and/or observed sporadically with no dose relationship and were therefore not considered to be test item-related

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmological findings at the end of the treatment period in animals at 600 mg/kg/day. Animals at 100 and 300 mg/kg/day were therefore not examined and neither were recovery animals at the end of the treatment-free period.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on hematology parameters.
Any differences between control and test item-treated groups, including those that were statistically significant, were not dose-related, of minimal magnitude and/or consistent with normal biological variations, and were therefore not considered to be test item-related.
Of note, the platelet count of female 3509 (300 mg/kg/day) at the end of the treatment period was particularly low (171 G/L versus mean platelet count of 970 G/L in control females) whereas no platelet aggregates were observed in the sample or on the blood smear. The hematology data of female 3509 were therefore not excluded from the mean calculation but in absence of correlating clinical signs or changes in associated parameters, this change was considered artefactual.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period and when compared to controls, dose-related decreases in total bilirubin and total protein concentrations, due to decreases in albumin, were observed in females at ≥ 100 mg/kg/day. These findings were considered to be attributed to the test item. They were all reversible at the end of the treatment-free period and were not considered adverse given their low magnitude.
All other variations in blood biochemistry parameters, including those that were statistically significant, were of low magnitude, observed with no dose relationship, noted in only a few animals and/or were consistent with normal biological variations, and were therefore considered to be unrelated to the test item. Specifically, the higher urea and/or creatinine concentrations in males at ≥ 100 mg/kg/day were not considered to be test item-related despite the histological changes observed in the kidneys because they were of low magnitude, poorly dose-related and consistent with biological variations.
Individual blood biochemistry data of males 3005 and 3006 at the end of the treatment period were excluded from mean calculations (due to aberrantly high potassium / low calcium concentrations suggesting a contamination of the samples with the anticoagulant K2EDTA used for hematology investigations). Somewhat high potassium / low calcium concentrations were also observed in male 1007 and females 2504 and 3505 at the end of the treatment period but as they were not considered to be aberrant and in absence of proved contamination, the individual blood biochemistry data of these females were not excluded from mean calculations.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on qualitative urinary parameters.
At the end of the treatment period and when compared to controls, the urinary volume was higher in males at 600 mg/kg/day and females at ≥ 300 mg/kg/day, correlating with slightly lower specific gravity. The urinary pH was slightly lower in males at ≥ 300 mg/kg/day. These findings were attributed to the test item. They were reversible at the end of the treatment-free period, although a slightly lower pH was still observed in males, and were not considered adverse given their slight magnitude.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A dose related minimal to moderate increase in liver weight was observed at all dose levels in both sexes. It correlated with macroscopic evidence of enlargement and/or accentuated lobular pattern, and microscopically with centrilobular hypertrophy and/or periportal vacuolation.
Moderate increase in kidney weight was observed in males from the dose of 300 mg/kg/day. It correlated at necropsy with various combinations of discoloration, irregular surface or color, and/or enlargement, and microscopically with an increase in hyaline droplets (positive for A2UG), granular casts and tubular basophilia.
Finally, minimal increases in thyroid weights were observed in males at 600 mg/kg/day, correlating microscopically with follicular cell hypertrophy, and in females at 100 and 300 mg/kg/day, but not at the highest dose of 600 mg/kg/day.
All other differences between test item-treated groups and controls were interpreted as incidental and/or within normal range of variation because of absence of dose relationship, because they lacked a histological correlate, or because the difference was very small.

At the End of the Treatment-Free Period
At the end of a 4-week treatment-free period, kidney and liver weights were minimally increased in male rats previously administered the test item at 600 mg/kg/day. However, the increases were smaller compared to previous time point, indicating ongoing recovery. There were no differences in thyroid weights between controls and animals previously administered the test item, indicating full recovery. Other differences in organ weights were considered fortuitous.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the End of the Treatment Period
Various combinations of accentuated lobular pattern, enlargement and/or irregular color were observed in the liver in both sexes and at all dose levels.
Various combinations of tan discoloration, irregular surface or color and enlargement were observed in the kidney from the dose of 300 mg/kg/day in males.
All other macroscopic observations were spontaneous in nature and bore no relationship to treatment.
At the End of the Treatment-Free Period
None of the macroscopic observations bore any relationship to treatment with the test item.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related findings during the FOB assessment with comparable clinical observations, reactivity to manipulation or different stimuli, and motor activity between control and test item-treated animals.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the End of the Treatment Period
There were treatment-related changes in the liver from both sexes, kidney and thyroid glands in males only, and stomach in females only.
Dose related minimal to marked increase in hyaline droplets in the kidneys was diagnosed in males at all doses. Hyaline droplets are composed of A2UG, and this was confirmed by Immuno Histo Chemistry (IHC) at the high-dose. The average score for IHC positivity in high dose rats was 3.9, vs. 2.0 in controls, confirming the increase noted in hematoxylin and eosin-stained slides. Hyaline droplets were associated with granular casts and tubular basophilia at all doses. Together, these findings, which were directly related to the test item, are part of the so called alpha 2u-globulin nephropathy, which is specific to male rats and irrelevant to human risk assessment (Swenberg, 1993). Nevertheless, in the context of this study, given the severity and the presence of hyaline casts, it was considered adverse at all dose levels.
Dose-related minimal to moderate centrilobular hypertrophy in the liver was observed at all doses in both sexes. Centrilobular hepatocellular hypertrophy in the liver is often due to induction of metabolic enzymes and as such it is a direct effect of the test item. It is well established as an adaptive and non-adverse change in the absence (as in this study) of histologic or clinical pathology alterations indicative of liver degenerative changes (Hall et al, 2012). It was accompanied by periportal vacuolation of hepatocytes, particularly in females. In the absence of accompanying clinical pathology changes, this was considered non-adverse. It was also accompanied by minimal follicular cell hypertrophy of the thyroid in males from the dose of 300 mg/kg/day. Thyroid hormones remained unaffected. Follicular cell hypertrophy in the thyroid often accompanies hepatic centrilobular hypertrophy in rats (Zabka et al, 2011), but this is not relevant to humans because of much shorter T4 half-life in rats (Capen, 1996).
Finally, inflammatory changes were observed in the forestomach in females at the high-dose, comprising acanthosis, mucosal hyperplasia and submucosal subacute inflammation. These changes are most probably related to small mucosal ulcerations, which were not on the plane of section. They were considered secondary to treatment, most probably stress-related (Everds et al, 2013).
All other changes were considered background.

At the End of the Treatment-Free Period
As shown in the table below, at the end of the treatment-free period, slight to moderate tubular basophilia and minimal or slight granular casts were observed only in males (a single occurrence of minimal focal tubular basophilia in one female is a background change, as observed at the end of treatment in control females). The IHC positivity for A2UG was comparable between control and treated males, with no increase in hyaline droplets observed with HE staining. Overall, the severity of the renal changes was lower, indicating ongoing recovery.
Similarly, only 1/5 males previously administered the test item had minimal centrilobular hypertrophy in the liver, indicating almost complete recovery.
Other test item related changes at the end of treatment were not observed, indicating full recovery.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Coaguation
There were no test item-related changes in prothrombin time at the end of the treatment period.
The statistically significantly shortened prothrombin time in males at 300 mg/kg/day compared to controls was considered to be incidental in absence of dose relationship.
Sperm parameters
There were no test item-related effects on sperm parameters at the treatment period, with comparable sperm motility and morphology, epididymal and testicular sperm counts between control and test item-treated males.

Details on results:

At the End of the Treatment Period
Various combinations of accentuated lobular pattern, enlargement and/or irregular color were observed in the liver in both sexes and at all dose levels.
Various combinations of tan discoloration, irregular surface or color and enlargement were observed in the kidney from the dose of 300 mg/kg/day in males. All other macroscopic observations were spontaneous in nature and bore no relationship to treatment.

At the End of the Treatment-Free Period
None of the macroscopic observations bore any relationship to treatment with the test item.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: absence of any adverse findings

Remarks on result:

other: due to adverse renal findings, no No Observed Adverse Effect Level (NOAEL) was established in male rats

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Dose Formulation Analysis

The test item concentrations in the administered dose formulations analyzed at the beginning and end of treatment period and in Week 6 remained within an acceptable range of -8.8% to +7.5% when compared to the nominal values (meeting the acceptance criterion of ± 10% of the nominal concentrations).
No test item was observed in the control dose formulations.

Applicant's summary and conclusion

Conclusions:

The oral administration of Isononyl isononanoate to rats at doses of 100, 300 or 600 mg/kg/day for 13 weeks, still clinically well tolerated, resulted in α2u-globulin nephropathy at all doses in males, with tubular basophilia and granular casts, which was considered adverse, although irrelevant for human risk assessment. It also induced hepatic centrilobular hypertrophy and periportal vacuolation at all doses and in both sexes, likely related to enzyme-inducing properties of the test item, accompanied by thyroid follicular cell hypertrophy (high-dose males only), and inflammatory changes in the forestomach in high-dose females, which were all considered non-adverse.
Consequently, due to adverse renal findings, no No Observed Adverse Effect Level (NOAEL) was established in male rats, while in absence of any adverse findings, the NOAEL was set at 600 mg/kg/day in female rats.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item, Isononyl isononanoate, following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals in the control and high-dose groups were held for a 4-week treatment-free period in order to evaluate the reversibility of any findings.
The study design was as follows:

Group	Number of animals	Animal number	Dose level (mg/kg/day)	Concentration (mg/mL)
1	15 males 15 females	1001-1015 1501-1515	0	0
2	10 males 10 females	2001-2010 2501-2510	100	20
3	10 males 10 females	3001-3010 3501-3510	300	60
4	15 males 15 females	4001-4015 4501-4515	600	120

The following parameters and end points were evaluated in this study: survival, clinical signs, functional observation battery, body weights, body weight gains, food consumption, ophthalmology, clinical pathology (hematology, coagulation, blood biochemistry, and urinalysis as well as thyroid hormones), estrous cycle, sperm parameters, gross necropsy findings, organ weights, and histopathologic examinations.
The actual test item concentrations in the analyzed dose formulations were -8.8% to +7.5% of nominal concentrations, meeting the acceptance criterion (± 10%).
There were no test item-related deaths during the study, no test item-related findings at the FOB assessment, no effects on body weight, despite several occurrences of slightly higher food consumption in both males and females at 600 mg/kg/day, no effects on ophthalmology, hematology or coagulation parameters, thyroid hormones, estrous cycle or sperm parameters.
Test item-related clinical signs consisted of reversible hypersalivation in a few males at ≥ 300 mg/kg/day and in a few females at 600 mg/kg/day. Changes at clinical pathology included reversible dose-related minimal decreases in total bilirubin and total protein (due to decrease in albumin) concentrations in females at ≥ 100 mg/kg/day and reversible higher urinary volume in males at 600 mg/kg/day and females at ≥ 300 mg/kg/day, with slightly lower urinary pH in males at ≥ 300 mg/kg/day.
Test item-related microscopic observations at the end of the treatment period included adverse changes in the kidneys in males at ≥ 100 mg/kg/day (hyaline droplets composed of α2u-globulin, associated with granular casts and tubular basophilia, correlating with increased kidney weights and various combinations of macroscopic tan discoloration, irregular surface or color and enlargement at ≥ 300 mg/kg/day), and non-adverse changes in the liver in males and females at ≥ 100 mg/kg/day (centrilobular hepatocellular hypertrophy, accompanied by periportal vacuolation of hepatocytes, and correlating with increased liver weights and macroscopic enlargement, irregular color and/or accentuated lobular pattern), in the thyroid in males at ≥ 300 mg/kg/day (follicular cell hypertrophy, correlating with increased thyroid weights at 600 mg/kg/day) and in the forestomach in females at 600 mg/kg/day (acanthosis, mucosal hyperplasia and submucosal subacute inflammation). At the end of the 4-week treatment-free period, full recovery was observed for thyroid and stomach changes, almost full recovery for hepatic changes, and ongoing recovery for renal changes.
In conclusion, the oral administration of Isononyl isononanoate to rats at doses of 100, 300 or 600 mg/kg/day for 13 weeks, still clinically well tolerated, resulted in α2u-globulin nephropathy at all doses in males, with tubular basophilia and granular casts, which was considered adverse, although irrelevant for human risk assessment. It also induced hepatic centrilobular hypertrophy and periportal vacuolation at all doses and in both sexes, likely related to enzyme-inducing properties of the test item, accompanied by thyroid follicular cell hypertrophy (high-dose males only), and inflammatory changes in the forestomach in high-dose females, which were all considered non-adverse.
Consequently, due to adverse renal findings, no No Observed Adverse Effect Level (NOAEL) was established in male rats, while in absence of any adverse findings, the NOAEL was set at 600 mg/kg/day in female rats.