Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Treatment commenced 12 August 2019 Experimental completion date (Pathology) 06 March 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 002

Cas Number:

58394-64-2

Constituent 2

Reference substance name:

Reference substance 003

Cas Number:

103-23-1

Constituent 3

Reference substance name:

Reference substance 001

Cas Number:

2451-84-5

Test material form:

liquid

Remarks:

Clear colorless liquid

Details on test material:

Batch No.: CH180307A

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies and as foreseen in the test guideline. The Sprague-Dawley (Crl:CD® (SD) IGS BR) strain was used because of the historical control data available at this laboratory and as this strain has been used in previous toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain/Species Crl:CD® (SD) IGS BR rat.
Supplier Charles River (UK) Ltd.
Number of animals 45 males and 45 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization 12 days before commencement of treatment.
Age of the animals at start of treatment 41 to 47 days.
Weight range of the animals at the start of treatment Males: 201 to 254 g
Females: 151 to 195 g
3.3.2 Allocation and Identification
Allocation Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.
3.3.3 Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed 20% of the mean for the appropriate sex. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment commenced
Body weight range extreme One female

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated; 15 air changes /hour.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.
Alarm systems Activated on ventilation failure and when temperature/ humidity limits exceeded.

Animal Accommodation
Cages Polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Cage distribution Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage Three or four of the same sex.
Bedding Softwood based bark-free fiber bedding, sterilized by autoclaving, which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen gnawing material A soft white untreated wood block; provided to each cage throughout the study (except during overnight for urine collection) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet Teklad 2014C pelleted Diet.
Availability Non-restricted (removed overnight before blood sampling for hematology, blood chemistry and during the period of urine collection).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted (except during the period of urine collection).

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the wood based bedding and Aspen gnawing material.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was chosen as requested by ECHA (Decision number CCH D-2114448639-34-01/F).

Vehicle:

arachis oil

Details on oral exposure:

Route Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg bw/day.
Volume dose 4 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as the treated groups.
Frequency Once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.


Formulation
Correction factor None.
Vehicle Arachis oil BP.

Method of preparation The required amount of test item was weighed into a suitable container. Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the test item and was magnetically stirred until uniformly mixed. A further amount of vehicle was added to make up to the required volume and further mixing, using a magnetic stirrer, was performed until the formulation was homogeneous. The remaining concentrations were then formulated in ascending order of concentration.

Frequency of preparation Weekly.
Storage of formulation Refrigerated (2 to 8°C).
Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed. There were no discrepancies.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis
Stability Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 250 mg/mL (Covance Study Number SP63HG) and at 3.70 to 251 mg/mL (Envigo Study Number 41500056) were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. These investigations confirmed the following:

24 hours stability at ambient temperature (15 to 25°C) at 1 to 250 mg/mL.

16 days stability at refrigerated temperature (2 to 8°C) at 3.70 to 251 mg/mL.

Achieved concentration Samples of each formulation prepared for administration in Weeks 1, 6 and 12 of treatment were analyzed for achieved concentration of the test item.

Analysis

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 males/ 10 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
The doses used in this study (0, 100, 300 and 1000 mg/kg bw/day) were selected in conjunction with the Sponsor.

A dose of 1000 mg/kg bw/day was considered a suitable high dose for use in this study based on the results of a four-week toxicity study in rats (Bayer Pharma AG Study Number T100005-6), at doses of 100, 300 and 1000 mg/kg bw/day. In that study, changes were seen in males in the liver at 300 mg/kg bw/day and in the kidneys at 100 mg/kg bw/day. The changes in the liver were considered an adaptive response to treatment with a xenobiotic and therefore was considered non-adverse. The changes in the kidneys were regarded to be adverse in the male (alpha-2u-globulin-mediated nephropathy). Therefore the no observed adverse effect level (NOAEL) for repeated oral administration of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in the four week study was 1000 mg/kg bw/day for female Wistar rats, but a NOAEL could not be established in male Wistar rats. To assist in determining doses for the present study, an OECD 421 study (Envigo study Number: JD44CD) in Sprague-Dawley rats, treated at 250, 500 and 1000 mg/kg bw/day resulted in a NO(A)EL of 1000 mg/kg bw/day. It was accepted that in that screening study, no hematology or blood chemistry investigations were required, and only limited histopathology evaluations were performed.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

Clinical Observations and Sensory Reactivity/Functional Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Immediately before dosing (Pre-dose).
At the end of dosing each group.
One to two hours after completion of dosing all groups.
As late as possible in the working day.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

The following measurements, reflexes and responses were recorded:
Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed for each animal.

At any point during the observations, additional comments were made as free text where considered appropriate.

Motor Activity
During Week 12 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Covance.

Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Body Weight

The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.
More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored. These data are retained in the study data but are not reported.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

Water Consumption
Fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not performed.

Ophthalmic Examination
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows:

Occasion Animals
Pretreatment All animals (including spares)
Week 12 All animals of Groups 1 and 4

Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food and prior to dosing. Sampling was performed on the morning after overnight collection of urine; therefore the animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasion:

Occasion Animals
Week 13 All animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:

Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected after overnight withdrawal of food and prior to dosing. Sampling was performed on the morning after overnight collection of urine; therefore the animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasion:

Occasion Animals
Week 13 All animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of:

Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acid (Bi Ac)
Urea*
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
High density phospholipid (HDL)
Low density phospholipid (LDL)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.
*Numerically equivalent to blood urea nitrogen (BUN)

Urinalysis
Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight at the following occasion:
Occasion Animals
Week 13 All animals

The individual samples were examined for the following characteristics:

Using manual methods:
Appearance (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips interpreted using the Clinitek®500 instrument (semi-quantitative method):
Glucose (Gluc)
Ketones (Keto)
Bile pigments (Bili)
Blood pigments (UBld)

Using a Cobas 6000 Analyzer (quantitative automated method):
Protein (T-Prot)
Sodium (T-Na)
Potassium (T-K)
Chloride (T-Cl)
Glucose (T-Gluc)
Creatinine (T-Creat)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.

Epithelial cells (Epi)
Leucocytes (WBC)
Erythrocytes (RBC)
Casts
Other abnormal components seen in the sample (A)

The slide was also examined for abnormalities in spermatozoa and crystals.

If there was insufficient sample to analyze all the parameters listed above, the following priority list applied:
Clarity
Colour
Volume
pH
Specific gravity
Clinitek list
Roche Cobas 6000 Analyzer list
Microscopy
3.6.9 Thyroid Hormone Analysis
Blood samples were collected at the following occasion:
Occasion Animals
At termination All animals

Parameters Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)
Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, samples were collected to allow satisfactory inter-group comparison.
Conditions No overnight deprivation of food.
Blood sample site Sublingual vein.
Anesthetic Isoflurane. The animals were not allowed to recover from the anesthesia.
Anticoagulant None.
Blood tube Minicollect tubes with clotting activator.
Blood volume 1.0 mL.
Temporary storage conditions Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000 g for 10 minutes at 4°C.
Separation of serum All available serum was transferred to an appropriately labelled polypropylene “cryo” tube using a plastic disposable pipette, then mixed by gentle 10-fold inversion. Following mixing, each serum sample was divided into two aliquots.
Aliquot volumes Aliquot 1: T3 and T4: 0.2 mL of serum
Aliquot 2: TSH: all remaining serum
Final storage conditions Deep frozen (approximately -60°C to -90°C).
Fate of samples Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.
T3 and T4 Performed by the Department of Bioanalysis, Covance.
TSH Performed by the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.

Vaginal Smears
Wet smears were taken as follows:
Wet smears Using pipette lavage for four days before scheduled termination.
Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus). These observations assisted in the histological evaluation of estrogen sensitive tissues.

Sacrifice and pathology:

Necropsy
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.
Schedule Animals were killed following 13 weeks of treatment (males 91 days; females 92 days).
Sequence To allow satisfactory inter-group comparison.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows:
Abnormalities
Adrenals
Aorta
Brain (cerebellum, cerebrum, midbrain)
Bone marrow smear
Cecum
Coagulating glands
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femur (femorotibial joint)
Head
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidney (IHC (males only, see Section 3.7)
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
- left axillary
Ovaries (with oviducts)
Pancreas
Pituitary
Prostate
Rectum
Tissue and regions examined

Salivary glands - submandibular
- sublingual
- parotid
Sciatic nerves
Seminal vesicles
Skin with mammary glands (inguinal area)
Skeletal muscle
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum (and bone marrow)
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina

Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment period.

Fixation Smears were air dried and subsequently fixed in methanol.
Analysis No examinations were performed, however, the smears were retained for possible future examination.
Retention The smears were transferred to archives and will be retained for the same period as the study raw data.

Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:

Kidneys Male animals for immunohistochemistry were fixed for 24 to 72 hours.
Testes In modified Davidson’s fluid.
Eyes In Davidson’s fluid prior to transfer to 70% industrial methylated spirit.
Bone marrow smears: See above.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List Animals of Groups 1 and 4 killed at the end of the treatment period.
Left kidney (all males) Fixed in 10% neutral buffered formalin for 24 to 72 hours prior to processing to wax blocks. Fixation time was standardized between groups; where possible.
Liver and abnormalities only Animals of Groups 2 and 3 killed at the end of the treatment period.
Routine staining Sections were stained with hematoxylin and eosin.

Immunohistochemistry (IHC)
Left kidney All terminal male animals from all groups.
Sections (4-5µ) were stained for alpha-2u-globulin using a validated immunohistochemistry method.

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Scheduled kill All animals of Groups 1 and 4. All specified in Section 3.7.
All animals of Groups 2 and 3. Liver and abnormalities only.
All males of Groups 2 and 3. Kidney.

Testes A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Statistics:

Please refer to "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

It was considered that the appearance and behavior of the animals were unaffected by treatment and there were no deaths during the treatment period.

One male (No. 13) that received 1000 mg/kg bw/day was recorded as underactive during Week 1 (Pre-treatment) and during the first two weeks of treatment. One male (No. 11) receiving 1000 mg/kg bw/day showed chromadacryorrhea from Week 4 until termination. One Control animal, one receiving 100 mg/kg bw/day, four receiving 300 mg/kg bw/day and three receiving 1000 mg/kg bw/day showed irritable behaviour, with or without vocalisation that was attributed to normal behaviour, demonstrated in this strain of rat. One Control male (No. 29) showed increased activity and noisy and rapid respiration in Week 4.

The majority of observations were evident for a short period and the incidences did not increase as treatment progressed; therefore the signs were considered not to be toxicologically significant.
PLEASE REFER TO ATTACHED TABLE.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study period

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall body weight gains in males receiving 300 or 1000 mg/kg bw/day was marginally low (85% or 87% of Control, respectively), without relationship to dose. This was largely attributed to low individual weight gains shown by three males at each dose (300 mg/kg bw/day No’s 33, 36 and 40 or 1000 mg/kg bw/day No’s 11, 15 and 17; that were otherwise overtly normal (in-life and post-life)) and, as there was no relationship to dose, this was considered not to be toxicologically significant.

Overall body weight gains of males and females receiving 100 mg/kg bw/day and females receiving 300 or 1000 mg/kg bw/day were unaffected by treatment. PLEASE REFER TO ATTACHED TABLE.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day. PLEASE REFER TO ATTACHED TABLE.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmoscopic findings that were attributable to treatment at 100, 300 or 1000 mg/kg bw/day.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination in Week 13 revealed no toxicologically significant differences from Control. All differences from Control were confined to one sex, were minor or lacked dose relationship and were therefore attributed to normal biological variation. These differences comprised:

At 1000 mg/kg bw/day and when compared with Control, large unstained cell count was high in males and in females (150% or 157%, respectively). In males, hematocrit was marginally high (106%) and in females, reticulocyte count and red cell distribution width were low (71% and 97%, respectively), monocyte count was high (157%) and activated partial thromboplastin time was short (70%) and neutrophil count was low, with an inverse relationship to dose at 100, 300 or 1000 mg/kg bw/day (60%, 63% or 69%), but overall white cell count was unaffected.
PLEASE REFER TO ATTACHED TABLE.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of the plasma in Week 13 revealed no toxicologically significant differences from Control. Differences from Control were largely confined to one sex, were minor or lacked dose relationship and were therefore attributed to normal biological variation. These differences comprised:

At 1000 mg/kg bw/day, aspartate amino-transferase activity in males and females was high (113% and 186%, respectively) and alkaline phosphatase activity was high (133%) in males only. In males and females at 1000 mg/kg bw/day respectively, urea and bile acid concentrations were high (males 136% and 259%; females 118% and 445%); however, the majority of individual bile acid concentrations were within the Control range. In females only, creatinine concentration was low (90%) and triglyceride concentration was high (157%) and glucose concentrations were high at 300 or 1000 mg/kg bw/day (117% or 119%, respectively).

There were minor differences from Control in the concentrations of all measured electrolytes in females receiving 1000 mg/kg bw/day (sodium (99%), potassium (128%), calcium (96%), phosphorus (87%)) and chloride concentrations were low in males and females at 1000 mg/kg bw/day (both 98%).
PLEASE REFER TO ATTACHED TABLE.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Examination of the urine in Week 13 revealed no toxicologically significant differences from Control. Total protein output was high in males and females receiving 1000 mg/kg bw/day (193% and 146% of Control, respectively) and pH was marginally low in males and females receiving 300 or 1000 mg/kg bw/day (males both 91%; females 89% or 87% of Control, respectively). Specific gravity was marginally high in females receiving 1000 mg/kg bw/day (101%). Urinary volume was high in males, compared with Control. PLEASE REFER TO ATTACHED TABLE.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Sensory Reactivity
Sensory reactivity in Week 12 was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Grip Strength
Grip strength in Week 12 was considered unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Females given 1000 mg/kg bw/day showed statistically significantly low hindlimb grip strength when compared to Control, with differences attributable to two females with atypically low grip strength values. All group mean values were within the Historical Control Data range, and in the absence of a similar decease in forelimb grip strength in this group of females, the differences from Control in hindlimb grip strength were considered fortuitous and unrelated to treatment.

Motor Activity
Motor activity in Week 12 was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.
PLEASE REFER TO ATTACHED TABLES.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The analysis of organ weights after 13 weeks of treatment indicated, when compared to Control, high adjusted kidney and liver weights in males and females receiving 1000 mg/kg bw/day (males 110% and 122%; females 106% and 115%, respectively). Adjusted thymus weight was low in males treated at 1000 mg/kg bw/day (82%).

PLEASE REFER TO ATTACHED TABLE.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed after 13 weeks of treatment revealed no test item related lesions. PLEASE REFER TO ATTACHED TABLE.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidney
Minimal to slight increased eosinophilic inclusions were present in the kidneys of four males treated at 100 mg/kg bw/day, three males treated at 300 mg/kg bw/day and five males treated at 1000 mg/kg bw/day.

Minimal to moderate increased IHC positive staining was observed in the kidneys stained for alpha-2u-globulin using a validated immunohistochemistry method in one control male, four males treated at 100 mg/kg bw/day or 300 mg/kg bw/day and nine males treated at 1000 mg/kg bw/day.

Liver
Minimal centrilobular hypertrophy was present in three males and five females treated at 1000 mg/kg bw/day.

Incidental Findings
The incidence and distribution of all other findings were considered to be unrelated to treatment.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

PLEASE REFER TO "ANY OTHERINFORMATION ON RESULTS" AND ATTACHED TABLE

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Estrous Cycles
There was no effect of treatment on estrous cycles at 100, 300 or 1000 mg/kg bw/day. PLEASE REFER TO ATTACHED TABLE.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

2

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

other: Renal

Organ:

kidney

Any other information on results incl. tables

All tables of the full study report are attached to "Attached Background Material".

 

Formulation Analysis

The mean concentrations of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in formulations analyzed during the study were within the applied limit of +10/-15% (-3.1 to -13.1%) of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis.

 

Samples for Week 6 were injected as per the analytical method. However due to poor chromatography only 1 of the 3 peaks could be quantified. The samples were reinjected outside of the stability period, but the linearity in this run failed and the results could not be reported.

 

Analyzed Concentrations for 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in Arachis Oil

Occasion	Group	Nominal concentration (mg/mL)	Analyzed concentration (mg/mL)			RME (%)	Difference from mean (%)
			Analysis 1	Analysis 2	Mean
Week 1	1	0	ND	ND	-	-	-
	2	25	23.7	24.2	24.0	-4.0	±1.07
	3	75	71.4	71.9	71.7	-4.4	±0.34
	4	250	241	243	242	-3.2	±0.35
Week 12	1	0	ND	ND	-	-	-
	2	25	23.2	22	22.6	-9.6	±2.80
	3	75	65.3	65.2	65.2	-13.1	±0.09
	4	250	219	222	221	-11.6	±0.78

RME     Relative mean error, representing the deviation from nominal

ND         Not detected

 

Thyroid Analysis

Meanserum triiodothyronine (T3) concentrationsat the end of the treatment period, at 100,300 or 1000 mg/kg bw/day, wereunaffected by treatment.

 

Mean serumthyroxine (T4)concentrations in females receiving 300 or 1000 mg/kg bw/day were marginally, but statistically significantly higher than Control (127% or 128%, respectively)at the end of the treatment period.

 

Mean serumthyroxineT4 concentrations in males receiving100,300 or 1000 mg/kg bw/dayand in females receiving 100 mg/kg bw/daywereunaffected at the end of the treatment period.

 

Mean serum thyroid stimulating hormone (TSH) concentrations at the end of the treatment period, at 100, 300 or 1000 mg/kg bw/day, were unaffected by treatment.

 

Summary of thyroid hormone results at the end of the treatment period

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Triiodothyronine (pg/mL)	599	609	623	605	872	860	938	910
Thyroxine (pg/mL)	48200	52300	50300	44200	41500	47400	52900*	53100*
Thyroid Stimulating Hormone(pg/mL)	1070	1370	1030	1270	718	732	683	675

^*-p<0.01

 

Summary of treatment related findings in the left kidney for males killed after 13 weeks of treatment

Group/sex		1M		2M		3M		4M
Dose (mg/kg bw/day)		0		100		300		1000
Increased Eosinophilic Inclusions
Minimal		0		4		2		5
Slight		0		0		1		0
Total		0		4		3		5
Increased IHC Positive Staining
Minimal		1		2		3		3
Slight		0		2		0		4
Moderate		0		0		1		2
Total		1		4		4		9
Number of tissues examined		10		10		10		10

 

Summary of treatment related findings in the liver for animals killed after 13 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Hypertrophy, Centrilobular
Minimal	0	0	0	3	0	0	0	5
Total	0	0	0	3	0	0	0	5
Number of tissues examined	10	10	10	10	10	10	10	10

Applicant's summary and conclusion

Conclusions:

It is concluded that oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8), to Sprague-Dawley rats for 13 weeks at doses up to 1000 mg/kg bw /day resulted in non adverse changes in the kidney (eosinophilic inclusions) of males treated at 100, 300 or 1000 mg/kg bw/day and non adverse changes in the liver (minimal centrilobular hypertrophy, attributed to high doses of a xenobiotic) of males and females treated at 1000 mg/kg bw/day. As the changes were non-adverse, the no observed-adverse-effect level (NOAEL) in this study was 1000 mg/kg bw/day.

Executive summary:

Summary

The objective of this study was to assess the systemic toxic potential ofreaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905‑983-8), when administered orally by gavage to Sprague‑Dawley rats for 13 weeks. This study was requested by the European Chemicals Agency (ECHA) Decision number: CCH-D-2114448639-34-01/F.

 

Three groups, each comprising ten males and ten females, received 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' at doses of 100, 300 or 1000 mg/kg bw/day, at a volume dose of 4 mL/kg bw. A similarly constituted control group received the vehicle (Arachis oil BP) at the same dose volume.

 

During the study, thyroid hormone analyses, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

 

Results

The appearance and behaviour of the animals were considered unaffected by treatment and there were no treatment-related findings at the sensory activity, grip strength and motor activity assessment in Week 12 and there were no deaths.

 

A marginal reduction in overall body weight gain was evident in males only at 300 or 1000 mg/kg bw/day, but there was no relationship to dose and this was therefore considered not to be toxicologically significant.

 

There were no effects of treatment on ophthalmoscopic findings, or estrous cycles.

 

There was no microscopic correlate for the minor differences in the cellular composition of the blood or blood plasma or in the urine and they weretherefore considered non‑adverse.

 

Serum T3, T4 and TSH concentrations were considered to be unaffected by treatment.

 

Increased eosinophilic inclusions and an increase in alpha-2u-globulin were evident in the kidneys of males treated at 100, 300 or1000 mg/kg bw/day, but there was no relationship to dose. At 1000 mg/kg bw/day, thiscorrelated with an increase inkidney weight. In the absence ofalpha-2u-globulin nephropathy, this findingwas considered non‑adverse.

 

Minimal centrilobular hypertrophy in the liver was evident in males and females treated at 1000 mg/kg bw/dayandcorrelated with an increase inliver weights. Thisfinding was attributed to an adaptive response, dueto metabolism of high doses of a xenobioticand wastherefore considered non‑adverse.

 

Conclusion

It is concluded that oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8), to Sprague-Dawley rats for 13 weeks at doses up to 1000 mg/kg bw /day resulted in non‑adverse changes in the kidney (eosinophilic inclusions) of males treated at 100, 300 or 1000 mg/kg bw/day and non‑adverse changes in the liver (minimal centrilobular hypertrophy, attributed to high doses of a xenobiotic) of males and females treated at 1000 mg/kg bw/day. As the changes were non-adverse, the no‑observed-adverse-effect level (NOAEL) in this study was 1000 mg/kg bw/day.