Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 25 November 2016 and 28 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Revised 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: GL500
Batch: 20130510
Purity: 99.81%
Expiry date: 10 May 2018
Storage Conditions: room temperature in the dark
Specific details on test material used for the study:

Test item identity (including alternative names) LGflex GL500, GL500, GL520
CAS number 1571954-81-8
Intended use Plasticizer
Appearance Clear colorless liquid
Storage conditions At ambient temperature (10 to 30C) and protected from light (although wasused and formulated in light)
Supplier Sponsor
Batch (Lot) number GLFG160607(only GLFG on test item containers)
Expiry date 1 June 2017
Purity 99.8%
Supplier’s responsibilities Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST strain
Sex:
male/female
Details on test animals and environmental conditions:
Animal Information
Animals
Strain/Species RccHan™:WIST rat.
Supplier Envigo RMS Limited.
Number of animals 45males and 45females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization 15 days before commencement of treatment.
Age of animals at start of treatment 44 to 50 days.
Weight range of the animals at the start of treatment Males: 140to 194g Females: 123to 154g.

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
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 minimizee ntry of external biological and chemical agents.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºCand 40-70%.
There was one deviation from these ranges, when a low temperature of 19”Cwas recorded on one day. Since this deviation was minor and of short durationit was not considered to have influenced the health of the animals and/or the outcome of the study.
Lighting Artificial lighting, 12 hours light : 12hours 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 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 Five 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 hematologyor blood chemistry).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
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 5 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 Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
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.
Vehicle:
corn oil
Details on oral exposure:
The study consisted of one control and three treated groups: 100, 300 and 1000 mg/kg/day.

Administration
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 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled bodyweight.
Control (Group 1) Vehicle at the same volume dose as the treated groups.
Frequency Once daily at approximately the same time each day.
Formulation Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Procedure
The samples were analyzed in accordance with the validated Envigo Analytical Procedure Method No.DFA/M116/16.
The analytical method involved extraction in 100% Acetoneand dilution in acetonitrile/water 90/10 v/v followed by reverse phase high performance liquid chromatographic analysis with ultra violet detection at 241 nm. Sample concentrations were determined with reference to external standards prepared in the concentration range Low µg/mlLto High µg/ml.
The standards used were prepared under this study and concurrent study SJ14LX.

Concentration of Dose Formulations
The formulations for Week 1, Week 4 and Week 13 for all groups were sampled, 1 × 10mL (accurately weighed), from the middle of the formulation by Pharmacy personnel.
Each sample was analyzed in duplicate in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

RESULTS AND CONCLUSION
The mean concentrations of GL500 in corn oil were within 4% of the nominal concentration, confirming the accuracy of formulation. The % difference from mean was within 2% confirming precise formulation.
The mean concentrations of GL500 in formulations prepared for administration in Week 1, 4 and 13 were between -1.5% and +3.7% of the nominal concentrations. These were within the applied limits of -15%/+10% of the nominal concentrations, demonstrating accurate formulation.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Calculated from the most recently recorded scheduled bodyweight
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Calculated from the most recently recorded scheduled bodyweight
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Calculated from the most recently recorded scheduled bodyweight
No. of animals per sex per dose:
10 per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The purpose of this study was to assess the systemic toxic potential of GL500 (a plasticizer) when administered orally, by gavage, to Han Wistar rats for 13 weeks.
Animal Model
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.
Route of Administration
The oral route of administration was chosen to simulate the conditions of potential human exposure. The test item was administered by gavage.
Rationale for Dose Level Selection
The doses used in this study (0, 100, 300 and 1000 mg/kg/day) were selected in conjunction with the Sponsor.
The dose levels were based on the results of a 2-week preliminary toxicity study in the Han Wistar rat at doses of 250, 500 and 1000 mg/kg/day (Envigo Study No. LD61QW). In this study there were no deaths, no adverse clinical signs and no effects on body weight gain or food consumption. Necropsy only revealed slightly high liver weights for males and females at 1000 mg/kg/day.
Consequently the high dose for this study was selected as 1000 mg/kg/day as this is the limit dose required by the OECD test guidelines. The low and intermediate dose levels of 100 and 300 mg/kg/day were chosen to allow the determination of any dose response and to establish a no-observed-adverse-effect-level (NOAEL).
Positive control:
Not relevant

Examinations

Observations and examinations performed and frequency:
Serial Observations

Clinical 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 ofnature 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 and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation.
One to two hours after completion of dosing of all groups.
As late as possible in the working day.

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.

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 significant difference from controlswas observed and consequently quantitative measurements were not performed. .

Ophthalmic Examination
The eyes of the animals were examined by means of a binocular indirect ophthalmoscopeas 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 from all animals after overnight withdrawal of food and prior to dosing in Week 13.

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collectedinto tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobinconcentration (Hb)
Erythrocyte count(RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin(MCH)* Mean cell hemoglobinconcentration (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 andappropriate 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 from all animals after overnight withdrawal of food and prior to dosing in Week 13.
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) Bile acids (Bi Ac)
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)
* Numerically equivalent to blood urea nitrogen (BUN)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzedalbumin concentration

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. 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 head until it was close to the nose (but not touching the whiskers). The 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 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 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 (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.




Sacrifice and pathology:
Terminal Investigations
Method of Kill
Carbon dioxide asphyxiation with subsequent exsanguination.

Necropsy
All animals were killed following 13weeks of treatment in a sequence to allow satisfactory inter-group comparison. 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.

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

Adrenals Ovaries
Brain Spleen
Epididymis Testis
Heart (including auricular and ventricular regions Thymus
Kidneys Uterus with cervix
Liver (section from 2 lobes)

Histology
Abnormalities
Adrenals Ovaries
Brain Pancreas
Caecum Pituitary
Colon Prostate
Duodenum Salivary glands (submandibular, sublingual, paratoid)
Epididymes Sciatic nerve
Esophagus Seminal vesicles
Eyes Skin with mammary glands (inguinal area)
Femur (with femoral joint and bone marrow) Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Head Spleen
Heart (including auricular and ventriaular regions) Sternum (and bone marrow)
Ileum (including Peyer’s patches) Stomach
Jejunum Testes
Kidneys Thymus
Liver (section of 2 lobes) Thyroid with Parathyroids
Lungs ( section from 2 major lobes with bronchi) Trachea
Lymph nodes (mesenteric and left axillary) Urinary bladder
Uterus (with cervix)
Vagina

Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for all animals killed at theend 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.
Kidneys, liver, thyroid glands, urinary bladder and abnormalities All animals of Groups 2 and 3 killed at the end of the treatment period.
Routine staining Sections were stained with hematoxylinand eosin.

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Scheduled kill All animals of Groups 1 and 4. All specified above.
All animals of Groups 2 and 3. Kidneys, liver, thyroid glands, urinarybladder and abnormalities.

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:
Data Evaluation
This report contains serial observations pertaining to all weeks of study completed, together with signs data collected during the necropsy period. In the case of detailed physical examination and arena observations, body weight, food consumptionand ophthalmic examination data, only information from the final week of the acclimatization period is presented.
Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices. It is, therefore, not always possible to derive exact group values from the data presented in the appendices.
Statistics:
See below

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on bodyweight gain.
Overall bodyweight gain was slightly low, when compared to the controls, at all doses in females but the extent of the differences from controls lacked dose-relationship and males were unaffected. The inter-group differences in females were therefore considered to represent normal biological variation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
A visual assessment of water intake did not reveal any clear effect of treatment
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related ophthalmoscopic findings
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematological investigation performed in Week 13 revealed, when compared to controls, slightly low hemoglobin concentrationin females receiving 1000 mg/kg/day which, in the absence of any effect on erythrocyte count, led to associated decreases of mean cell hemoglobin and mean cell volume in these animals. There were no similar findings in males.
All other inter-group differences from controls, including those that attained statistical significance, were minor, seenin one sex only or were without dose-relationship and were therefore considered to represent normal biological variation. Such differences included reductions of reticulocyte numbers in males receiving 300 or 1000 mg/kg/day since the extent of inter-group differences was minimal, there was no dose response and there was no associated change in erythrocyte numbers. They also included variations of clotting times (reduced activated partial thromboplastin times in males but, conversely, increased activated partial thromboplastin times in the females which also had some inconsistent variation of prothrombin times) since the extent of the differences from controls was minimal in each case and there was no dose response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma in during Week 13 of treatment revealed, when compared to the controls, slightly high creatinine concentrations in males receiving 1000mg/kg/day.
Triglyceride concentrations were slightly high, compared to controls, in males receiving 1000mg/kg/day but there was no associated change in cholesterol levels. There was also a small increase of triglyceride concentration in females receiving 1000 mg/kg/day but this was not statistically significant.
Plasma potassium concentrations were slightly low, when compared to the controls, in males receiving 1000 mg/kg/day; females were unaffected.
In males receiving 1000 mg/kg/day there was an increase in the albumin to globulin ratio which was attributed to a minimal increase in the albumin fraction and a minimal decrease in the globulin fraction. The extent of these trends, which were not evident in females, indicated that these findings wereconsidered to be of no toxicological significance.
All other inter-group differences from controls, including those that attained statistical significance, were minor, seen in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory Reactivityand Grip Strength
Sensory activity and grip strength were unaffected by treatment.

Motor Activity
Motor activity was unaffected by treatment
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The analysis of organ weights after 13 weeks of treatment revealed, when compared with the controls, high absolute and body weight-adjusted liver weights in females given 300mg/kg/day and in males and females given 1000 mg/kg/day. Absolute and body weight-adjusted kidney weights were also slightly high in females given 1000 mg/kg/day.
All other inter-group variations of organ weights were minor, lacked dose-relationship or were associated with differences in terminal bodyweight and were therefore attributed to normal biological variation
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed after 13 weeks of treatment did not reveal any changes related to GL500.
All macroscopic findings were of the type encountered normally in Han Wistar rats at these laboratories.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Premature Decedents
There were five premature decedents in male animals treated with 1000/750 mg/kg bw/day.
Animals 63, 64, 67 and 70 showed lymphoid depletion and thymic atrophy along with changes in the stomach, animal 70 also had adrenal gland hypertrophy.
Animal 65 showed lymphoid and bone marrow depletion, thymic atrophy and adrenal gland hypertrophy. These animals primarily showed signs of stomach and intestinal malfunction along with changes in lymphoid tissue and adrenal glands which are likely to be secondary and stress related. Reflux may have occurred in these animals but there was no obvious change in the tissues examined.

Terminal Kill
Stomach
Changes were noted in animals from all groups treated with the test item in the glandular stomach.
Diffuse inflammatory cell infiltration was present in three males treated with 10 mg/kg bw/day, five males treated with 100 mg/kg bw/day and all males treated with 1000/750 mg/kg bw/day and in two females treated with 10 mg/kg bw/day, two females treated with 100 mg/kg bw/day and seven females treated with 1000/750 mg/kg bw/day. The severity ranged from minimal to moderate and showed a dose dependent trend. Focal infiltration was present in a further two females treated with 1000/750 mg/kg bw/day.
Mucous cell hypertrophy was noted in one male treated with 10 mg/kg bw/day, three males treated with 100 mg/kg bw/day and four males treated with 1000/750 mg/kg bw/day and in one female treated with 10 mg/kg bw/day, four females treated with 100 mg/kg bw/day and eight females treated with 1000/750 mg/kg bw/day. The severity was minimal or mild and the change showed a dose dependent trend.
Foveolar hyperplasia, minimal (with eosinophilic globule cells) was present in two surviving males and five females treated with 1000/750 mg/kg bw/day.
Paneth cell metaplasia was present in all surviving males treated with 1000/750 mg/kg bw/day.
Erosion of the glandular mucosa was present in one male treated with 100 mg/kg bw/day and three males treated with 1000/750 mg/kg bw/day along with one female treated with 10 mg/kg bw/day and two females treated with 1000/750 mg/kg bw/day.
Changes were noted in the non-glandular region in animals treated with 100 or 1000/750 mg/kg bw/day.
Hyperplasia, focal or diffuse was present in two males treated with 100 mg/kg bw/day and two surviving males treated with 1000/750 mg/kg bw/day and in one female treated with 100 mg/kg bw/day and four females treated with 1000/750 mg/kg bw/day.
No other findings were present at histopathology which could be attributed to treatment with the test item or that were considered to be adverse in nature.
Thymus
Minimal atrophy was present in three females treated with 100 mg/kg bw/day and is likely to indicate a secondary stress-related effect and correlated with the reduction in organ weight noted at necropsy. As such, this is considered not to be adverse in nature.
No other findings were present at histopathology which could be attributed to treatment with the test item.
Salivary Gland
After examination of salivary gland from all animals considerable individual variation was noted and no changes were considered to be related to treatment.

Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with GL500 were seen in the liver and thyroid glands.
Liver
Diffuse centrilobular hypertrophy was recorded in the majority of males and a few females treated with 1000 mg/kg/day, with a higher severity and/or incidence occurring in the males.
Thyroid glands
Follicular cell hypertrophy was recorded in a fewmales and females treated with 300 or 1000mg/kg/day. This finding was also reported inacontrol male.
Incidental findings
The incidence and distribution of all other findings were considered to be unrelated to treatment.
Chronic cardiomyopathy was recorded in a few males treated with 1000 mg/kg/day but this was considered within the normal background pathology of Han Wistar rats. This finding was characterized by multifocal cardiomyocyte necrosis with inflammatory cell infiltration and was a morphologic manifestation of spontaneous rodent or murine progressive cardiomyopathy in young rodents (<3 months

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
urinalysis
behaviour (functional findings)
immunology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
It is concluded that oral administration of GL500 to Han Wistar rats for 13 weeks at doses up to 1000 mg/kg/day did not cause any toxicologically significant response to treatment. There was an adaptive response in the liver at 1000 mg/kg/day that resulted in a secondary, rodent-specific stimulation of the thyroid gland. The thyroid gland findings were also present at 300mg/kg/day. Since none of the findings in this studywas considered adverse, the no-observed-adverse-effect level (NOAEL) in this study was considered to be 1000mg/kg/day.
Executive summary:

The purpose of this study was to assess the systemic toxic potential of GL500 (a plasticizer) when administered orally to Han Wistar rats for 13weeks.  The control group received the vehicle (corn oil) at the same volume dose.  The study design was as follows:

 Group  Treatment  Dose (mg/kg/day)  Number of animals           
        Male  Female
 1  Control  0  10  10
 2  GL500  100  10  10
 3  GL500  300  10  10
 4  GL500  1000  10  10

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

Results

There were no deaths, the general appearance and behaviour of the animals were unaffected by treatment and sensory activity, grip strength and motor activity were also unaffected by treatment.

There was no effect of treatment on bodyweight gain or food and water consumption.

There were no treatment-related ophthalmoscopic findings.

The haematological investigation in Week13 revealed slightly low haemoglobin concentration, mean cell haemoglobin and mean cell volume in females receiving 1000mg/kg/day.

Biochemical changes in the blood plasma in Week 13 comprised: slightly high creatinine and triglyceride concentrations in males receiving 1000mg/kg/day; low potassium concentrations in males receiving 1000 mg/kg/day; slightly increased albumin to globulin ratio in males receiving 1000 mg/kg/day.

After 13 weeks the liver weights of females given 300mg/kg/day and of males and females given 1000 mg/kg/day were high, and kidney weights were also slightly high in females given 1000 mg/kg/day.

There were no treatment-related macroscopic findings.

The microscopic examination after 13 weeks of treatment revealed the presence of centrilobular hepatocellular hypertrophy in the liver of males and females given 1000mg/kg/day and follicular cell hypertrophy in the thyroid glands in males and females given 300 or 1000 mg/kg/day.

Conclusion

It is concluded that oral administration of GL500 to Han Wistar rats for 13 weeks at doses up to 1000 mg/kg/day did not cause any toxicologically significant response to treatment.  There was an adaptive response in the liver at 1000 mg/kg/day that resulted in a secondary, rodent-specific stimulation of the thyroid gland.  The thyroid gland findings were also present at 300mg/kg/day.  Since none of the findings in this studywas considered adverse, the no-observed-adverse-effect level (NOAEL) in this study wasconsidered to be 1000mg/kg/day.