Registration Dossier

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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 06 December 2017 Experimental completion date: 06 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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

Constituent 1
Chemical structure
Reference substance name:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
EC Number:
276-014-8
EC Name:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
Cas Number:
71786-60-2
Molecular formula:
Not applicable
IUPAC Name:
2,2'-(C12-18 evennumbered alkyl imino) diethanol
Test material form:
liquid
Details on test material:
- Chemical name: Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
- CAS number: 276-014-8

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant.
Specific details on test material used for the study:
Test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives

2,2’-(C12-18 even-numbered alkyl imino)diethanol.
Bis(2-hydroxyethyl)cocoalkylamine
CAS number 71786-60-2
Intended use Substance used in industry.
Appearance Light yellow liquid.
Storage conditions At ambient temperature (15 to 25C), in the dark.
Supplier Sponsor.
Batch number 1373142
Expiry date 28 November 2018
Purity 98%
Supplier’s responsibilities Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample A 0.5 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan™;WIST ) strain was used because of the historical control data available at this laboratory
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals
Strain/Species
RccHan™;WIST rat.

Supplier
Envigo RMS Limited.

Number of animals
44 males and 44 females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization 12 days before commencement of treatment.

Age of animals at start of treatment 41 to 47 days.

Weight range of the animals at the start of treatment Males: 124 g to 175 g
Females: 115 g to 145 g

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.

Animal Replacement
Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

Replacement before treatment commenced
Ocular abnormalities One female

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. No replacements were necessary.

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.

Temperature and relative humidity
Monitored and maintained within the range of 20-24ºC and 40-70%.

There was one minor deviation from the range (19ºC on Day 67). This deviation was minor and of short duration and was not considered to have influenced the health of the animals and/or the outcome of the study.

Lighting
Artificial lighting, 12 hours light : 12 hours dark.

Electricity supply
Public supply with automatic stand-by generators.

Animal Accommodation
Cages
Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.

Cage distribution
Males and females were blocked by group 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
Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen chew block Provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet Teklad 2014C Diet.
Availability Non-restricted (removed overnight before blood sampling for hematology or 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).

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily by oral gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
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/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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Liquid formulation
The homogeneity and stability of formulations during refrigerated storage (2 to 8°C) was confirmed and supplied by the Sponsor (Harlan Project Nos. 0142-0416 and 0142 0417) however, no documented stability during ambient temperature was included as part of these trials. Therefore, a trial to assess the stability and homogeneity of the test item in the liquid matrix at ambient temperate (15 to 25°C) for up to four hours was conducted as part of this study.



Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2.5 and 250 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.

Achieved concentration
Samples of each formulation prepared for administration in Weeks 1 and 13 of treatment were analyzed for achieved concentration of the test item.

Preparation of Calibration Standards
A primary standard solution (500000 ng/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives in propan-2-ol (100 mL). A econdary standard solution (10000 ng/mL) was prepared by appropriate dilution of the primary standard using diluent (10 mL). A tertiary standard solution (1000 ng/mL) was prepared by appropriate dilution of the secondary standard using diluent to the final volume of 10 mL.

Solutions for instrument calibration were prepared by appropriate dilution of the tertiary standard using diluent and contained Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives at nominal concentrations of 10 ng/mL, 20 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 80 ng/mL and 100 ng/mL.

Calibration solutions were injected onto the LC-MS/MS, at the beginning of each sample analysis sequence, using the conditions detailed in the chromatographic section. The standard prepared at 50 ng/mL was injected in duplicate to bracket every three samples.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in a suitable volume of propan-2-ol. The extract was then diluted further with propan-2-ol with final dilutions performed using diluent, to provide a solution containing Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives at an expected concentration within the range 20 ng/mL to 80 ng/mL.

The concentration of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives in the final solution was quantified by LCMS/MS as detailed in the chromatographic section.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (arachis oil) with known amounts of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives. The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Instrumentation Parameters
High performance liquid chromatograph and mass spectrometer (LC-MS/MS): Agilent 1100 pump, Perkin Elmer PE200 Autosampler and Sciex API 3000 equivalent mass spectrometer

Column: Agilent Poroshell SB C18, 2.7 μm, 100 x 4.6 mm

Column temperature: 45ºC

Sample temperature: Ambient

Mobile Phase A: 0.1M ammonium acetate(aq)/Acetic acid 100/0.1 v/v

Mobile Phase B: MeOH / Acetic Acid 100/0.1 v/v

Flow rate: 0.5 mL/minute

Ionisation: Turboionspray – positive ion mode

Source temperature: +500°C

Collision gas: Nitrogen

Collision energy: 35 eV

Dwell time: 100 ms

Pause time: 5 ms

Ions to be monitored: m/z 274.5 to m/z 106.1 (used for quantitate)

Run Time: 12 minutes

Approximate Retention time: 8.1 minutes

Calculations
The peak counts response for Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area counts of the peak observed at the characteristic retention time for Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives in sample and procedural recovery chromatograms was measured. The concentration of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was determined using the following equation:

Determine the response factor for the single standard.

Response factor (RF) = Calibration standard peak response (Ac ) / Concentration of calibration standard (ng/mL)

Then for each sample chromatogram determine the concentration of the test item using the following equations:

Analysed concentration (mg/mL) = (As/RF) x (V/W) x (D/1,000,000)

Procedural recovery values were determined using the following equation:

Procedural recovery = (Analyzed concentration/ Fortified concentration) x 100

Sample concentrations were corrected for procedural recoveries using the following equation:
Corrected concentration, mg/mL = Analysed concentration, mg/mL x 100 /R

Where
Ac = Peak response for test item in single standard.
As = Peak response for test item in sample chromatogram.
RF = Mean response factor of appropriate set of bracketing standard
V = Dilution volume (mL)
W = Sample weight (g)
R = Appropriate procedural recovery value
D = Density of sample (g/mL)
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control - vehicle only
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
125 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:
The doses used in this study (0, 10, 30 and 125 mg/kg/day) were selected in conjunction with the Sponsor.

Dose levels were selected for this study, based on the results of a 14-day dose range finding study (Harlan Project No. 0142-0416) and a combined repeat dose toxicity study with production/developmental toxicity screening test repeated dose study (Harlan Project No. 0142-0417).

In the 14-day dose range finding study all animals given 250 mg/kg/day were sacrificed on Day 10 due to adverse clinical signs such as lethargy, hunched posture, dehydration and diarrhea and there was also significant body weight loss, increased water intake and reduced food consumption. After 14 days at 150 mg/kg/day there were no adverse clinical signs or body weight effects but the macroscopic investigations revealed increased liver weight and thickening of the non-glandular region of the stomach.

Based on these findings a high dose level of 125 mg/kg/day was selected for use on the reproduction/developmental toxicity screening test repeated dose study. In that study there were no adverse clinical signs and no effects on body weight or food consumption, however slightly increased liver and spleen weights were observed at macroscopic examination. At microscopic examination, acanthosis was seen in the fore-stomach of males and females treated at 125 mg/kg/day. Since the forestomach present in the rodent is not present in the human stomach and the functionally of the stomach was not compromised, these findings are not considered to be indicative of a risk to human health.

Consequently, 125 mg/kg/day was selected as the high dose level in this 90-day study. The intermediate and low dose levels of 30 and 10 mg/kg/day, respectively, were selected to allow evaluation of any dose related trends and to determine the no-observed-adverse-effect level.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Serial Observations
Clinical and Behavioral 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 treatment period, detailed observations were recorded at the following times in relation to dose administration:

Daily during Week 1 of treatment Pre-dose observation. One to two hours after completion of dosing of all groups. As late as possible in the working day.

Daily from Week 2 of treatment Pre-dose observation. One to two hours after completion of dosing of all groups.

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 who was 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 probe/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.
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 Envigo.

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; consequently quantitative measurements were not performed.

Ophthalmic Examination
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope (at the discretion of the examining veterinary surgeon a slit-lamp biomicroscope was also used) as follows:
Occasion Animals
Pretreatment All animals
Week 12 All animals of Groups 1 and 4
Week 13 All animals of Groups 2 and 3

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 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 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 P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea*
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
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)

Urine Collection
Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight at the following occasions:
Occasion Animals
Week 4 All animals
Week 13 All animals

The samples were collected and stored deep frozen (-60 to -90C) before dispatch. The samples were dispatched to the Responsible Scientist on dry ice.
The results of the analysis of the urine samples will be reported separately and are not part of this study.
Sacrifice and pathology:
Terminal Investigations

Method of Kill
Carbon dioxide asphyxiation with subsequent exsanguination.

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.

Sequence To allow satisfactory inter-group comparison.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows:

Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Abnormalities * * *
Adrenals * * * *
Aorta * * *
Brain (cerebellum, cerebrum, midbrain) * * * *
Cecum * * *
Colon * * *
Duodenum * * *
Epididymides * * * *
Esophagus * * *
Eyes * * *
Femur (femorotibial joint) * † †
Head * # #
Heart (including auricular and ventricular regions) * * * *
Ileum * * *
Jejunum * * *
Kidneys * * * *
Liver (section from two lobes) * * * *
Lungs (section from two major lobes including bronchi) * * *
Lymph nodes - mesenteric * * *
- left axillary * * *
Ovaries * * * *
Pancreas * * *
Pituitary * * *
Prostate * * *
Salivary glands - submandibular * † †
- sublingual * † †
- parotid * † †
Sciatic nerves * † †
Seminal vesicles * * *
Skin with mammary glands (inguinal area) * * *
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 * * *
* Organs weighed, samples fixed or sections examined microscopically.
# Not examined.
† Only one examined.
Animal ID retained

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at the end of the treatment period.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes In modified Davidson’s fluid.
Eyes In Davidson’s fluid.

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 All animals of Groups 1 and 4 killed at the end of the treatment period.
Abnormalities, eyes, liver, thyroid and stomach only All animals of Groups 2 and 3 killed at the end of the treatment period.
Routine staining Sections were stained with hematoxylin and eosin.

Light Microscopy
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.
Other examinations:
Serial Observations
Clinical Observations
Signs are considered in two parts: detailed observations recorded at times in relation to dose administration, classified as ‘signs associated with dosing’, and extended changes in condition, classified as ‘detailed physical examination and arena observations’.
Clinical observations are presented, providing detail of type of sign, day or week of occurrence and information on the duration of the sign applicable.
Body Weight
Group mean weight changes were calculated from the weight changes of individual animals.
Food Consumption
Group mean food consumptions and standard deviations for each period were derived from unrounded cage values. Overall mean food consumption values were calculated for each cage and the mean of these cage means were calculated for each group/sex.
Hematology, Peripheral Blood
The abbreviation used has the following meaning:
INS Insufficient sample

Blood Chemistry
Albumin to globulin ratio (A/G Ratio) was calculated as:
A/G Ratio = Albumin concentration
Total protein – albumin concentration

Blood urea nitrogen is numerically equal to, and is derived from urea since the results are expressed as mmol/L.
3.8.2 Terminal Investigations
Organ Weights
Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.
Pathology
The abbreviations used have the following meanings:
Lt Left
GALT Gut associated lymphoid tissue
Statistics:
Please refer to "Any other information on materials and methods incl. tables"

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
The general appearance and behaviour of animals receiving 10 or 30 mg/kg/day and the behaviour of animals receiving 125 mg/kg/day were unaffected by treatment and no deaths occurred during the study.
Mortality:
no mortality observed
Description (incidence):
No deaths occured during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The overall weight gain of males receiving 30 or 125 mg/kg/day were slightly lower than the controls (5 and 6% reduction, respectively) but statistical significance was not attained and, consequently, these trends were attributed to normal biological variation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
The ophthalmic examination in Week 12 indicated the presence of opacification/cataract in the lens of seven males and seven females receiving 125 mg/kg/day. In the majority of affected animals this presented as slight opacification/cataract on the posterior capsule but in two males and two females was a seen as a total nuclear cataract that was bilateral in one male and one female and unilateral in one male and one female. The presence of the total nuclear cataract obscured examination of the vitreous and fundus but in animals where these structures could be assessed there was no evidence for any treatment-related change.
In view of the presence of the lenticular findings, the ophthalmoscopic examination was extended to males and females treated at 10 or 30 mg/kg/day (examination performed in Week 13), there were no treatment-related findings at these doses.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematology, Peripheral Blood
The haematological examination in Week 13 indicated low haematocrit, haemoglobin concentration and mean cell volume and a marginal increase of red cell distribution width in females receiving 125 mg/kg/day. There was also a small reduction of mean cell volume in females receiving 10 or 30 mg/kg/day but this was unlikely to be of any toxicological significance in the absence of any change in the erythrocyte indices at these doses. With the exception of low mean cell volume, these trends were not evident in males. In males there was a marginal increase in erythrocyte count and decreased mean cell volume at 30 or 125 mg/kg/day and low mean cell haemoglobin at 125 mg/kg/day but these were considered of no toxicological significance, particularly as haemoglobin concentration was unaffected.
Males receiving 30 mg/kg/day and males and females receiving 125 mg/kg/day showed an increase, compared to controls, of neutrophil count, with the extent of the effect in males being dose-related, and lymphocyte counts were high in females receiving 125 mg/kg/day. In addition, at 125 mg/kg/day, there were also increases of eosinophil count in males, basophil and large unstained cell count in females and monocyte count in both sexes but the these differences from controls were minor. As a consequence of these findings, the total leucocyte counts of animals receiving 125 mg/kg/day were higher than controls.

All other inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the slightly but statistically significantly shorter activated partial thromboplastin time in males receiving 125 mg/kg/day since reduced clotting times are considered of no toxicological significance, prothrombin times were unaffected and there was no similar finding in females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood Chemistry
The biochemical examination of the blood plasma in Week 13 indicated, when compared to controls, high urea concentrations in males and females receiving 125 mg/kg/day which, in females, associated with low plasma creatinine concentration.

The plasma glucose concentrations of males receiving 125 mg/kg/day were slightly higher than controls, but there was no similar finding in females.

In females treated at 125 mg/kg/day there was an increase of total plasma cholesterol concentration but there was no effect on triglyceride concentration and males were unaffected.

Calcium concentrations were increased, compared to controls, in females treated at 30 or 125 mg/kg/day but there was no similar finding in males.

Total protein was increased, compared to controls, in females treated at 125 mg/kg/day. This was attributed to an increase in both the albumin and globulin fractions and led to a decrease of the albumin to globulin ratio. There was also a small reduction of the albumin to globulin ratios of females receiving 30 mg/kg/day. Males were unaffected.

All other inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the high aspartate amino transferase activities in males receiving 10 mg/kg/day since this was due to high values in four animals (with values >100 U/L) and there was no similar finding at higher doses. They also included the statistically significantly low total bilirubin concentrations in females receiving 125 mg/kg/day since none of the individual values was abnormal and reduced bilirubin concentrations are of no toxicological importance.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength were unaffected by treatment.

Motor Activity
Motor activity was unaffected by treatment.

Statistical significance was attained in the males receiving 10 mg/kg/day for an increase of low (cage-floor activity) beam breaks at the 6 minute interval and for the increase of high (rearing) beam breaks at the 24 minute interval in females receiving 125 mg/kg/day. These were transient variations that were confined to one sex and. in the absence of any alteration of total beam breaks, were attributed to normal variation.
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 controls, high body weight-adjusted adrenal gland weight in males given 125 mg/kg/day, high body weight-adjusted kidney weight in males and females given 125 mg/kg/day, and high body weight-adjusted liver weight in females given 30 mg/kg/day and in males and females given 125 mg/kg/day.

All other inter-group differences from control were minor, lacked dose-relationship or (where applicable) were confined to one sex and were therefore attributed to normal biological variation.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macropathology
The macroscopic examination performed after 13 weeks of treatment revealed the following changes in the stomach and eyes related to the test item.

Stomach
Thickened nonglandular stomach mucosa was seen in all males treated at 30 or 125 mg/kg/day and in females treated at 10 (1 animal), 30 (7 animals) or 125 (all animals) mg/kg/day.

Eye
Unilateral or bilateral lens opacity was observed in a few animals treated at 125 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings
Changes related to treatment with Ethanol, 2,2'-iminobis-, NC12-18-alkyl derives were seen in stomach (nonglandular stomach), liver, thyroid glands and eyes.

Nonglandular stomach
Epithelial hyperplasia (acanthosis) and hyperkeratosis was seen in animals treated with 30 or 125 mg/kg/day, with the extent of this finding being dose-related. A female given 10 mg/kg/day was also affected, to a minimal extent.

Thyroid glands
Hypertrophy of the follicular cells was observed in both sexes treated at 125 mg/kg/day.

Liver
Increased rarefaction of the hepatocytes was seen in both sexes treated at 30 or 125 mg/kg/day, with the incidence being higher in females than in males.

Eyes
Lens degeneration was observed in four males and three females given 125 mg/kg/day.

Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
ophthalmological examination
Key result
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
ophthalmological examination

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
eye
Organ:
lens
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

 Macropathology

Macroscopic examination performed after 13 weeks of treatment revealed the following changes in the stomach and eyes related to the test item.

Stomach

Thickened nonglandular stomach mucosa was seen in all males treated at 30 or 125mg/kg/dayand in females treated at 10 (1 animal), 30 (7 animals) or 125 (all animals)mg/kg/day.

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

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Thickened – Nonglandular Mucosa

0

0

10

10

0

1

7

10

Number of animals examined

10

10

10

10

10

10

10

10

Eye

Unilateral or bilateral lens opacity was observed in a few animals treated at 125 mg/kg/day.

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

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Opaque

0

0

0

3

0

0

0

2

Number of animals examined

10

10

10

10

10

10

10

10

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

  Histopathology

Treatment-related findings

Changes related to treatment with Ethanol, 2,2'-iminobis-, NC12-18-alkyl derives were seen in stomach (nonglandular stomach), liver, thyroid glands and eyes.

Nonglandular stomach

Epithelial hyperplasia (acanthosis) and hyperkeratosis was seen in animals treated with 30 or 125 mg/kg/day, with the extent of this finding being dose-related. A female given 10 mg/kg/day was also affected, to a minimal extent.

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

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Epithelial hyperplasia (acanthosis)

 

 

 

 

 

 

 

 

Minimal

0

0

1

1

0

1

2

0

Slight

0

0

3

1

0

1

7

1

Moderate

0

0

6

6

0

0

0

9

Marked

0

0

0

2

0

0

0

0

Total

0

0

10

10

0

2

9

10

Hyperkeratosis

 

 

 

 

 

 

 

 

Minimal

0

0

1

1

0

1

4

0

Slight

0

0

6

1

0

0

5

1

Moderate

0

0

2

8

0

0

0

9

Total

0

0

9

10

0

1

9

10

Number of tissues examined

10

10

10

10

10

10

10

10

Thyroid glands

Hypertrophy of the follicular cells was observed in both sexes treated at 125 mg/kg/day.

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

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Hypertrophy, Follicular cells

 

 

 

 

 

 

 

 

Minimal

0

0

0

4

0

0

0

5

Slight

0

0

0

3

0

0

0

2

Total

0

0

0

7

0

0

0

7

Number of tissues examined

10

10

10

10

10

10

10

10

Liver

Increased rarefaction of the hepatocytes was seen in both sexes treated at30 or125 mg/kg/day, with the incidence being higher in females than in males.

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/day)

0

10

30

125

0

10

30

125

Rarefaction, Increased

 

 

 

 

 

 

 

 

Minimal

0

0

0

2

0

0

1

7

Slight

0

0

0

1

0

0

0

0

Total

0

0

0

3

0

0

1

7

Number of tissues examined

10

10

10

10

10

10

10

10

Eyes

Lens degeneration was observed in four males and three females given125 mg/kg/day.

 

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

 

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Degeneration, Lens

 

 

 

 

 

 

 

 

Minimal

0

0

0

1

0

0

0

1

Slight

0

0

0

1

0

0

0

2

Moderate

0

0

0

2

0

0

0

0

Total

0

0

0

4

0

0

0

3

Number of tissues examined

10

10

10

10

10

10

10

10

Incidental findings

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

Attached

TABLES  

TABLE 1 JH45BW 90-day rat study Detailed physical examination and arena observations - group distribution of observations      

                               

TABLE 2 JH45BW 90 day rat study Sensory reactivity observations and grip strength - summary of findings during Week 12 of treatment   

       

TABLE 3 JH45BW 90 day rat study Motor activity - group mean scores (beam breaks) during Week 12 of treatment  

                   

TABLE 4 JH45BW 90 day rat study Body weight - group mean values (g)      

                            

TABLE 5 JH45BW 90 day rat study Food consumption - group mean values (g_animal_week)        

    

TABLE 6 JH45BW 90 day rat study Ophthalmic examination - group distribution of observations before commencement of treatment and during Week 12 or 13

TABLE 7 JH45BW 90 day rat study Hematology - group mean values during Week 13 of treatment

TABLE 8 JH45BW 90 day rat study Blood chemistry - group mean values during Week 13 of treatment

TABLE 9 JH45BW 90 day rat study Organ weights - group mean absolute and adjusted values (g) for animals killed after 13 weeks of treatment

TABLE 10 JH45BW 90 day rat study Macropathology - group distribution of findings for animals after 13 weeks of treatment

TABLE 11 JH45BW 90 day rat study Histopathology - group distribution of findings for animals after 13 weeks of treatment                                                          

 

FIGURES   

                                                                                                                                 

FIGURE 1 JH45BW 90 day rat study Motor activity - group mean scores (beam breaks) for males and females during Week 12 of treatment            

                                     

FIGURE 2JH45BW 90 day rat study Body weight - group mean values (g) for males and females

APPENDIX

Results Envigo Study Number JH45BW 90 day rat study Formulation Analysis results

 

Applicant's summary and conclusion

Conclusions:
It is concluded that oral administration of Ethanol, 2,2'-iminobis , NC12-18-alkyl derives (a substance used in industry) to Han Wistar rats for 13 weeks caused adverse findings in the eyes at 125 mg/kg/day (lenticular opacification/cataract and degeneration) and stomach at 30 and 125 mg/kg/day (epithelial hyperplasia (acanthosis) and hyperkeratosis). The findings in the nonglandular stomach are considered to be due to local irritant effects of the test formulations. There were also rodent-specific findings in the thyroid glands (follicular cell hypertrophy), that was likely secondary to induction of liver enzymes, and a minor and non adverse effect on hepatocellular glycogen (increased rarefaction). In view of the presence of adverse findings in the eyes at 125 mg/kg/day the systemic no-observed-adverse-effect level (NOAEL) in this study was considered to be 30 mg/kg/day.
Executive summary:

 Summary

The purpose of this study was to assess the systemic toxic potential of Ethanol, 2,2'‑iminobis‑, NC12-18-alkyl derives (a substance used in industry) when administered orally, by gavage, to Han Wistar rats for 13 weeks. Three groups, each comprising ten males and ten females, received the test material at doses of 10, 30 or 125 mg/kg/day and a similarly constituted control group received the vehicle (arachis oil)at the same volume dose. 

During the study, 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, organ weight, macropathology and histopathology investigations were undertaken.

Results

The general behavior, sensory reactivity responses, grip strength and motor activity were not affected by treatment, no deaths occurred during this study and there was no effect of treatment on body weight gain or on food and water consumption.

Opaque eyes were observed from Week 9 in three males and two females receiving 125 mg/kg/day. In most cases this was initially a unilateral finding that rapidly became bilateral as the treatment period progressed. Animals receiving 10 or 30 mg/kg/day were unaffected.

The ophthalmic examination in Week 12 indicated the presence of opacification/cataract in the lens of seven males and seven females receiving 125 mg/kg/day, located predominantly on the posterior capsule but in two males and two females there was a total nuclear cataract.

The haematological examination in Week 13 indicated low haematocrit, haemoglobin concentration and mean cell volume and a marginal increase of red cell distribution width in females receiving 125 mg/kg/day, with low mean cell volume also occurring in males at this dose. Treatment-related alterations of leucocyte numbers included increased neutrophil count in males receiving 30 mg/kg/day and males and females receiving 125 mg/kg/day, increased lymphocyte counts in females receiving 125 mg/kg/day and minor increases at 125 mg/kg/day of eosinophil count in males, basophil and large unstained cell count in females and monocyte count in both sexes, leading to increased total leucocyte counts in animals receiving 125 mg/kg/day.

Biochemical changes in the blood plasma in Week 13 comprised: high urea concentrations in males and females receiving 125 mg/kg/day; low creatinine and high total cholesterol concentrations in females receiving 125 mg/kg/day; high glucose concentrations in males receiving 125 mg/kg/day; increased calcium concentrations in females treated at 30 or 125 mg/kg/day; high total protein concentration in females receiving 125 mg/kg/day, which was attributed to an increase in both the albumin and globulin fractions and led to a decrease of the albumin to globulin ratio. 

Organ weight analysis after 13 weeks of treatment indicated high adrenal gland weight in males given 125 mg/kg/day, high kidney weight in males and females given 125 mg/kg/day and high liver weight in females given 30 mg/kg/day and in males and females given 125 mg/kg/day.

The macroscopic examination performed after 13 weeks of treatment revealed thickening of the nonglandular mucosa of the stomach at 30 and 125 mg/kg/day, with one female at 10 mg/kg/day being similarly affected, and unilateral or bilateral lens opacity in three males and two females treated at 125 mg/kg/day.

Histopathological findings that were attributed to treatment occurred in the nonglandular stomach (epithelial hyperplasia (acanthosis) and hyperkeratosis at 10 (one female, minimal), 30 and 125 mg/kg/day, with the extent of this finding being dose-related), liver (increased hepatocyte rarefaction in animals, particularly females, given 125 mg/kg/day), thyroid glands (follicular cell hypertrophy at 125 mg/kg/day) and eyes (lenticular degeneration infour males and three females given125 mg/kg/day).

Conclusion

It is concluded that oral administration of Ethanol, 2,2'-iminobis‑, NC12-18-alkyl derives (a substance used in industry) to Han Wistar rats for 13 weeks caused adverse findings in the eyes at 125 mg/kg/day (lenticular opacification/cataract and degeneration) and stomach at 30 and 125 mg/kg/day (epithelial hyperplasia (acanthosis) and hyperkeratosis). The findings in the nonglandular stomach are considered to be due to local irritant effects of the test formulations. There were also rodent-specific findings in the thyroid glands (follicular cell hypertrophy), that was likely secondary to induction of liver enzymes, and a minor and non-adverse effect on hepatocellular glycogen (increased rarefaction). In view of the presence of adverse findings in the eyes at 125 mg/kg/day the systemic no-observed-adverse-effect level (NOAEL) in this study was considered to be 30 mg/kg/day.