Registration Dossier

Administrative data

Description of key information

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol was tested in an oral 90 -day repeated dose toxicity study according to OECD 408 guideline. In this study, at 1000/750 mg/kg bw/day, mortality prevents conclusion of a lack of effect on body function and therefore, excluding local irritancy and in terms of systemic toxicity only, it may be postulated that a NOAEL is 100 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 25 JUly 2016 and 04 April 2017
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification : Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol
Appearance: Beige solid flakes
Purity : UVCB 100%
Batch Number: AAE1455700
Label : Aradur 3380-1 CH Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, ogligomeric reaction producta with ethane-1,2-diol and glycerol Expire Date 10.10.2018 Net 1L
Date Received: 15 July 2016
Storage Conditions: Room temperature, in the dark
Expiry Date : 10 October 2018
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST strain
Sex:
male/female
Details on test animals and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nine days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 188 to 217g, the females weighed 135 to 162g, and were approximately six to eight weeks old.

Animal Care and Husbandry
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively.

IN-LIFE DATES: From: 16 September 2016 To: 16 December 2016
Route of administration:
oral: gavage
Details on route of administration:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP.
The test item was administered daily, for up to ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Vehicle:
arachis oil
Details on oral exposure:
Dose Administration
Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
Male Female
Control 0 4 0 10 (1-10) 10 (11-20)
Low 10 4 2.5 10 (21-30) 10 (31-40)
Intermediate 100 4 25 10 (41-50) 10 (51-60)
High 1000/750# 4 250/187.5# 10 (61-70) 10 (71-80)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.
The test item was administered daily, for up to ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatogrphy with UV detection (HPLC-UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Analytical Procedure
Preparation of Calibration Standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument parameters section.

Preparation of Test Samples
The formulations received were extracted with extract solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with extract solvent this was then ultra-sonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration. See Table 1 for preparation details.

Preparation of Accuracy and Precision Samples
This was performed under study number PD09LW.
The concentration of Test Item in the final solution was quantified by HPLC using UV detection as detailed in the instrument parameters section 2.2.6.

Instrumentation Parameters
HPLC : Agilent Technologies 1100 or 1200, incorporating autosampler and workstation
Column : Luna 5µ C18 (250 x 4.6 mm id)
Mobile phase : Acetonitrile
Flow-rate 1 mL/min
UV detector wavelength: 250 nm
Injection volume: 25µL
Retention time :~ 2.9 mins

Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.

Validation of the Analytical Procedure
This was performed under study number PD09LW. The data was found to have a linear correlation within the calibration range of 0.0553 mg/mL to 0.2212 mg/mL. The R² fit of the calibration curve to the data was 0.9977, and was considered to be acceptable.
Method accuracy (recovery) and precision were confirmed and the results indicated a mean recovery value of 99% (CV=2.23%, n=5) for 2.5 mg/mL and 96% (CV=3.46%, n=5) at 250 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

Homogeneity and Stability in Vehicle Formulations
This was performed under study number PD09LW. The homogeneity and stability of Test Item in Arachis Oil BP formulations was assessed with respect to the level of concentration at nominal concentrations of 2.5 mg/mL and 250 mg/mL(homogeneity), 25 mg/mL and 250 mg/mL (stability).
Homogeneity was confirmed. The mean analyzed concentration for the nine samples remained within 10% of the initial time zero value and the variation was less than 10%.

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved.

Results
Concentration of Dose Formulations
The mean concentrations of Test Item in test formulations analyzed during the study and the results are presented in Table 1, below. The mean concentrations were within applied limits ±20%, confirming accurate formulation (with the exception of analysis 5).

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision during study number PD09LW.
The homogeneity and stability was confirmed for Test Item in Arachis Oil BP formulations at nominal concentrations of 2.5 mg/mL and 250 mg/mL(homogeneity), 25 mg/mL and 250 mg/mL (stability) when stored for 4 hours during study number PD09LW.
The mean concentrations of Test Item in test formulations analyzed for the study were within ±20% of nominal concentrations, confirming accurate formulation (with the exception of analysis 5).

Duration of treatment / exposure:
90 days
Frequency of treatment:
Once Daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Reduced to 750.0 on Day 34 (males) or Day 33 (females)
No. of animals per sex per dose:
10 per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The study was performed according to the study plan and was designed to investigate the systemic toxicity of the test item, by repeated oral administration to the Wistar Han™: RccHan™: WIST strain rat for a period of up to ninety consecutive days at dose levels of 10, 100 and 1000 mg/kg bw/day. The dose level for the highest treatment group was reduced to 750 mg/kg bw/day on Day 34 (males) or Day 33 (females) due to the severity of clinical signs being noted and the fact that one male animal was humanely killed on animal welfare grounds on Day 32. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).
The dose levels were chosen in collaboration with the Sponsor and based on available toxicity data provided by the Sponsor. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Positive control:
Not relevant
Observations and examinations performed and frequency:
Serial Observations
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Specialist Evaluations

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all non-recovery animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each nonrecovery animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each non-recovery animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Ophthalmoscopic Examination
The eyes of all non-recovery control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye. Following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

Sacrifice and pathology:
Terminal Investigations
Necropsy
On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals Ovaries
Brain Spleen
Right Epididymis Right Testis
Heart Thymus
Kidneys Uterus
Liver

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint)• Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and pons) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymes ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes * and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including Peyer’s patches) Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Tongue•
Lungs (with bronchi)# Trachea
Lymph nodes (mandibular and mesenteric) Urinary bladder
Mammary gland Uterus (with cervix)
Muscle (skeletal) Vagina

All tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: J Schofield). All tissues from control and 1000/750 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of stomach (males and females), salivary glands (males) and thymus (females) from animals in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist. A peer review was conducted by the Responsible Scientist at the Test Site (Envigo CRS Limited, Huntingdon).

• Retained only and not processed
* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Other examinations:
Data Evaluation
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarized in tabular form.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Surviving animals treated with 1000/750 mg/kg bw/day exhibited the following clinical observations during the course of the study. Increased salivation was evident in all animals of either sex from Days 23 to 90. Instances of noisy respiration were noted in all surviving males and nine females from Day 22 (males) or Day 36 (females) to Day 91. Laboured respiration was noted in one male and two females which persisted in each animal for one day only (Day 50 male and Days 36 or 62 females). Decreased respiratory rate was noted in one female on Day 36 only and increased respiratory rate was noted in two females on either Day 62 or 71, hunched posture was noted in one female on Day 62. Red/brown staining around the snout was noted in one male on Day 86 for one day only.
Sporadic occurrences of increased salivation were noted in eight males and five female animals treated with 100 mg/kg bw/day from Days 24 to 90. Occasional instances of noisy respiration were noted in six males and three females treated with 100 mg/kg bw/day and in one male treated with 10 mg/kg bw/day from Days 63 to 89 or Day 80 respectively.
A mass under the right forelimb was also noted for male 26 on Days 89 to 91, however, as no other such observations were apparent in any other animal this was likely to be incidental and unrelated to treatment with the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
Five male animals treated with 1000/750 mg/kg bw/day were killed in extremis during the study from Days 32 to 71 (one male when treated at 1000 mg/kg bw/day and the other four animals when treated at 750 mg/kg bw/day). Prior to death, clinical signs noted for these animals generally included increased salivation, hunched posture, pilo-erection and decreased respiratory rate. Four of these animals exhibited laboured respiration, two exhibited a distended abdomen, two exhibited diarrhoea, three exhibited noisy respiration, one exhibited dehydration and noisy respiration and two animals exhibited gasping respiration.
Necropsy findings in these animals generally included gaseous distension throughout the gastrointestinal tract and stomach findings which included; thin non-glandular region and/or pale glandular region, one animal also exhibited a thin caecum and a raised limiting ridge in the stomach. Three animals exhibited discoloration to the lungs which included dark and/or pale areas. Other isolated findings included flaccid kidneys, dark liver, small prostate and seminal vesicles and a small spleen.
There were no further unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals treated with 1000/750 mg/kg bw/day showed a general reduction in body weight gain from Week 2 of the treatment period achieving statistical significance during Weeks 2, 3, 4, 5 and 7. Animals which were subsequently humanely killed exhibited actual body weight losses during Weeks 2, 4 and 10 (even with these values removed the mean body weight gains were still lower than control). Actual body weight losses were also evident in three surviving males during Week 10. A statistically significant increase was noted during Week 11 (even though one animal continued to exhibit a body weight loss) followed by mean body weight losses being noted during the final two weeks of the treatment period. An overall reduction in body weight gain of approximately 30% when compared to control was noted for these animals at the end of the treatment period.
Male animals treated with 100 mg/kg bw/day showed an initial reduction in body weight gain from Week 2 of treatment which achieved statistical significance during Week 3, however, recovery was evident over the next eight weeks as body weight gains were generally similar to control. As with the male animals treated with 1000/750 mg/kg bw/day a statistically significant increase in body weight gain was noted during Week 11, which was followed by mean body weight losses during Week 12. Reduced body weight gains were noted during Week 13 with approximately 50% of animals showing body weight losses on each occasion. An overall reduction in body weight gain of approximately 10% when compared to control was noted for these animals at the end of the treatment period.
No such effects were noted in male animals treated with 10 mg/kg bw/day or in any treated female
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Male animals treated with 1000/750 mg/kg bw/day showed a marginal reduction in food consumption during Weeks 4, 10, 12 and 13, a more marked reduction was noted during Week 5 which was considered to be due to one male animal which exhibited significant body weight loss during this time frame.
No such effects were detected in male animals treated with 10 or 100 mg/kg bw/day or in any treated female animals.
Any reductions/fluctuations in food conversion efficiencies in treated animals were considered likely to be due to fluctuations in body weight gains and/or food intake.
Food efficiency:
no effects observed
Description (incidence and severity):
Any reductions/fluctuations in food conversion efficiencies in treated animals were considered likely to be due to fluctuations in body weight gains and/or food intake.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no treatment-related effects on water consumption.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination of animals of both sexes from the control and 1000/750 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At Week 13 assessments, there were considered to be no adverse effects detected in the hematological parameters examined.
At Week 13 assessments, surviving male animals treated with 100 or 1000/750 mg/kg bw/day showed statistically significant decreases (p<0.05 - p<0.01) in mean corpuscular hemoglobin concentration and lymphocytes in a dose related manner. Male animals treated with 1000/750 mg/kg bw/day also showed statistically significant increases (p<0.01) in platelet counts and statistically significant decreases (p<0.05) in hemoglobin and mean corpuscular hemoglobin. For values that were shown to be decreased (excluding lymphocytes) when compared to control, one to four control animals actually exhibited values that were higher than the normal control data ranges which has accentuated the effect of lower values (from the historical control data ranges) noted in several of the treated male animals (in these parameters). The majority of values noted in relation to platelet counts in both the control and animals treated with 1000/750 mg/kg bw/day were within the normal historical control data ranges. As similar effects were not seen in the female animals and there were no histopathological correlates it is considered that these findings are spurious and cannot be attributed to the pathology noted in these animals and as such cannot be attributed to treatment with the test item. The reduction in lymphocytes (where all values for treated male animals were lower than the historical control data range) along with the slight reduction in thymus weights (when compared to control) may indicate a secondary stress-related effect and is considered to be non-adverse in nature.
Females treated with 100 or 1000/750 mg/kg bw/day showed statistically significant increases (p<0.05) in hemoglobin and hematocrit. With the exception of hematocrit where 50% of values were outside of the historical control data range for female animals treated with 1000/750 mg/kg bw/day the majority of values actually lay within these ranges. Therefore, in the absence of a true dose related response and as similar findings were not seen in the male animals and there were no histopathological correlates, these findings too are considered to be unassociated to treatment with the test item.
No such effects were detected in animals of either sex treated with 10 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At Week 13 assessments, there were considered to be no treatment-related effects detected in the blood parameters examined.
At Week 13 assessments, females treated with 1000/750 mg/kg bw/day showed a statistically significant increase (p<0.05) in creatinine, however, three control values were lower than the historical control data range and one value was higher, three of the treated female animals also exhibited values that were higher than the historical control data range. As there were no histopathological correlates and male animals didn't show a similar response this is considered to be due to normal biological variation and unrelated to treatment with the test item.
No such findings were noted in males treated with 1000/750 mg/kg bw/day or in animals of either sex treated with 10 or 100 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Behavioral Assessments
Behavioural assessments confirmed the clinical signs of noisy respiration in six male animals treated with 1000/750 mg/kg bw/day from Days 11 to 67, the majority of these findings were sporadic in nature, however, one male animal exhibited noisy respiration on each separate occasion from Day 18 to Day 67. One further male exhibited labored respiration on Day 11 and another male exhibited decreased respiratory rate, hunched posture and pilo-erection on Day 32 with one other male animal exhibiting signs of tip-toe gait on Days 53 and 60. Two female animals from this treatment group also exhibited noisy respiration on each occasion between Days 11 and 25. Noisy respiration was also confirmed in one male animal treated with 10 mg/kg bw/day on Day 53.
There were no treatment-related changes in the behavioural parameters measured for animals of either sex treated with 100 mg/kg bw/day or in female animals treated with 10 mg/kg bw/day.

Functional Performance Tests
There were no toxicologically significant changes in functional performance.
Overall activity was statistically significantly increased (p<0.05) across all female treatment groups, however, as these were not in a dose related manner and male animals didn’t show any similar effects these findings are considered to be incidental and unrelated to treatment with the test item
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were considered to be no adverse effects detected in the organ weights measured.
Male animals treated with 1000/750 mg/kg bw/day exhibited a statistically significant increase in adrenal weights (p<0.05) both absolute and relative to terminal body weight. Approximately 40% of control animals and all of the surviving treated males showed values that exceeded the normal control ranges. As there were no corresponding test item related microscopic findings seen in the adrenals or any histopathological correlates to account for the effects on these organ weights, these findings are considered to be of no toxicological significance.
Female animals treated with 1000/750 and 100 mg/kg bw/day exhibited a statistically significant reduction in thymus weights (p<0.01) both absolute and relative to terminal body weight. Approximately 30% of control animals showed values that were higher than the normal control ranges, whereas all treated females showed values that were within the historical control data range. These findings were considered to be in relation to a secondary stress response and as such these findings are considered to be non adverse.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Findings amongst animals that survived until terminal necropsy which were considered to be related to treatment were as follows:
Stomach - Glandular region - sloughing - One female treated with 10 mg/kg bw/day and one female treated with 1000/750 mg/kg bw/day.
Stomach - Non-glandular region - thin - one male treated with 1000/750 mg/kg bw/day.
Stomach - Glandular region - pale and/or thickened - two females treated with 10 mg/kg bw/day, one male and one female treated with 100 mg/kg bw/day, four males and nine females treated with 1000/750 mg/kg bw/day.
Stomach - Glandular region - red patches - two males treated with 100 mg/kg bw/day, four males and six females treated with 1000/750 mg/kg bw/day.
Stomach - Limiting Ridge - Raised - two males and one female treated with 100 mg/kg bw/day, four males and four females treated with 1000/750 mg/kg bw/day.
Stomach - Gaseous distention - one male treated with 1000/750 mg/kg bw/day.
Stomach - red patches - one male treated with 1000/750 mg/kg bw/day.
Salivary glands were enlarged in one male treated with 1000/750 mg/kg bw/day.
The following findings were noted at necropsy but were without histopathological correlates or true dose related responses and therefore were considered to be incidental and unrelated to treatment with the test item:
One male animal treated with 10 mg/kg bw/day exhibited a mass on the esophagus and a further mass which contained thick yellow/green fluid.
One control male and two control females, three females treated with 10 mg/kg bw/day, four females treated with 100 mg/kg bw/day and one female treated with 1000/750 mg/kg bw/day had reddened lungs at necropsy.
One female treated with 1000/750 mg/kg bw/day exhibited increased renal pelvic space in both kidneys.
One control male had mottled appearance of the kidneys.
One female treated with 10 mg/kg bw/day and two females treated with 1000/750 mg/kg bw/day exhibited dark patches on the lungs.
One female treated with 1000/750 mg/kg bw/day had red discolouration of the lymph nodes
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:
not specified
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Remarks:
In terms of systemic toxicity, treatment with dose levels up to 100 mg/kg bw/day showed that only local irritant effects were seen and that these changes were not sufficient to disrupt normal body functions. At 1000/750 mg/kg bw/day, mortality prevents conclusion of a lack of effect on body function and therefore, excluding local irritancy and in terms of systemic toxicity only, it may be postulated that a NOAEL is 100 mg/kg bw/day.
Key result
Critical effects observed:
not specified
Conclusions:
The oral administration of Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol to Wistar rats of both sexes at dose levels of 0, 10, 100 or 1000/750 mg/kg bw/day for a period for up to ninety consecutive days, resulted in the early termination of five male animals treated with 1000/750 mg/kg bw/day from Days 32 to 71 due to the severity of clinical signs noted and body weight losses. Histopathology revealed that 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.
Treatment related effects were also noted at histopathology examination in surviving animals of either sex across all treatment groups, which included changes in the glandular stomach. These findings generally showed dose dependent trends. There was mucosal cell hypertrophy, erosion and inflammatory cell infiltration indicating an adverse change at all dose levels. In addition, foveolar hyperplasia and paneth cell hyperplasia occurred in animals treated with 1000/750 mg/kg bw/day only.
A No Observed Adverse Effect Level (NOAEL) could, therefore, not be established due to the microscopic stomach changes evident in animals of either sex across all treatment groups. In terms of systemic toxicity, treatment with dose levels up to 100 mg/kg bw/day showed that only local irritant effects were seen and that these changes were not sufficient to disrupt normal body functions. At 1000/750 mg/kg bw/day, mortality prevents conclusion of a lack of effect on body function and therefore, excluding local irritancy and in terms of systemic toxicity only, it may be postulated that a NOAEL is 100 mg/kg bw/day.
Executive summary:

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guideline:

·        The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to ninety consecutive days, at dose levels of 10, 100 and 1000 mg/kg bw/day. The dose level for the highest treatment group was reduced to 750 mg/kg bw/day on Day 34 (males) or Day 33 (females) due to the severity of clinical signs being noted and the fact that one male animal was humanely killed on animal welfare grounds on Day 32. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from all control and high dose animals (Groups 1 and 4) as well as any gross lesions from Groups 1 to 4 was performed in the first instance. As there were possible treatment-related findings in the stomach, salivary glands and thymus, examination of these tissues was subsequently extended to include relevant animals from the low and intermediate dose groups.

Results….

Mortality

Five male animals treated with 1000/750 mg/kg bw/day were killedin extremisduring the study from Days 32 to 71 (one male when treated at 1000 mg/kg bw/day and the other four animals when treated at 750 mg/kg bw/day). Prior to death, clinical signs noted for these animals generally included increased salivation, hunched posture, pilo-erection and decreased respiratory rate. Four of these animals exhibited laboured respiration, two exhibited a distended abdomen, two exhibited diarrhoea, three exhibited noisy respiration, one exhibited dehydration and noisy respiration and two animals exhibited gasping respiration. 

Necropsy findings in these animals generally included gaseous distension throughout the gastrointestinal tract and stomach findings which included; thin non-glandular region and/or pale glandular region, one animal also exhibited a thin caecum and a raised limiting ridge in the stomach. Three animals exhibited discoloration to the lungs which included dark and/or pale areas. Other isolated findings included flaccid kidneys, dark liver, small prostate and seminal vesicles and a small spleen.

There were no further unscheduled deaths during the study.

Clinical Observations

Surviving animals treated with 1000/750 mg/kg bw/day exhibited the following clinical observations during the course of the study; there were frequent instances of increased salivation. Occasional instances of noisy respiration and hunched posture and isolated occurences of laboured respiration, decreased respiratory rate and increased respiratory rate and red/brown staining around the snout.  

Animals treated with 100 mg/kg bw/day exhibited sporadic occurrences of increased salivation and occasional instances of noisy respiration.

One male animal treated with 10 mg/kg bw/day exhibited noisy respiration for one day only. No such effects were evident in females treated with 10 mg/kg bw/day.

Behavioral Assessment

Behavioural assessments confirmed the clinical signs of noisy respiration in six male animals treated with 1000/750 mg/kg bw/day from Days 11 to 67. Isolated instances of laboured respiration were evident in one male, decreased respiratory rate, hunched posture and pilo-erection were evident in a further male and tip-toe gait was evident in one male. Two female animals from this treatment group also exhibited noisy respiration on two occasions. Noisy respiration was also confirmed in one male animal treated with 10 mg/kg bw/day on Day 53.

There were no treatment-related changes in the behavioural parameters measured for animals of either sex treated with 100 mg/kg bw/day or in female animals treated with 10 mg/kg bw/day.

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Male animals treated with 1000/750 mg/kg bw/day showed a general reduction in body weight gain from Week 2 of the treatment period. Mean body weight losses were noted during the final two weeks of the treatment period. An overall reduction in body weight gain of approximately 30% when compared to control was noted for these animals at the end of the treatment period.

Male animals treated with 100 mg/kg bw/day showed an initial reduction in body weight gain from Week 2 of treatment. However, recovery was evident after Week 3 as body weight gains were generally similar to control. As with the male animals treated with 1000/750 mg/kg bw/day mean body weight losses were noted during Week 12, reduced gains were then noted during Week 13 with approximately 50% of animals showing body weight losses on each occasion. An overall reduction in body weight gain of approximately 10% when compared to control was noted for these animals at the end of the treatment period. 

No such effects were noted in male animals treated with 10 mg/kg bw/day or in any treated female.

Food Consumption

Male animals treated with 1000/750 mg/kg bw/day showed a marginal reduction in food consumption during Weeks 4, 10, 12 and 13, a more marked reduction was noted during Week 5. 

No such effects were detected in male animals treated with 10 or 100 mg/kg bw/day or in any treated female animals.

Water Consumption

There were no treatment-related effects on water consumption.

Ophthalmoscopy

Ophthalmoscopic examination of animals of both sexes from the control and 1000/750 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

Hematology

At Week 13 assessments, there were considered to be no adverse effects detected in the hematological parameters examined.

Blood Chemistry

At Week 13 assessments, there were considered to be no treatment-related effects detected in the blood parameters examined.


Necropsy

Findings amongst animals that survived until terminal necropsy which were considered to be related to treatment were as follows:

Stomach - Glandular region - sloughing - One female treated with 10 mg/kg bw/day and one female treated with 1000/750 mg/kg bw/day.

Stomach - Non-glandular region - thin - one male treated with 1000/750 mg/kg bw/day.

Stomach - Glandular region - pale and/or thickened - two females treated with 10 mg/kg bw/day, one male and one female treated with 100 mg/kg bw/day, four males and nine females treated with 1000/750 mg/kg bw/day.

Stomach - Glandular region - red patches - two males treated with 100 mg/kg bw/day, four males and six females treated with 1000/750 mg/kg bw/day.

Stomach - Limiting Ridge - Raised - two males and one female treated with 100 mg/kg bw/day, four males and four females treated with 1000/750 mg/kg bw/day.

Stomach - Gaseous distention - one male treated with 1000/750 mg/kg bw/day.

Stomach - red patches - one male treated with 1000/750 mg/kg bw/day.

Salivary glands were enlarged in one male treated with 1000/750 mg/kg bw/day.

All other findings were considered to be incidental and unrelated to treatment with the test item.

Organ Weights

There were considered to be no adverse effects detected in the organ weights measured.

Histopathology

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.

Conclusion

The oral administration of Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol to Wistar rats of both sexes at dose levels of 0, 10, 100 or 1000/750 mg/kg bw/day for a period for up to ninety consecutive days, resulted in the early termination of five male animals treated with 1000/750 mg/kg bw/day from Days 32 to 71 due to the severity of clinical signs noted and body weight losses. Histopathology revealed that these animals primarily showed signs of stomach and intestinal

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1 GLP conform study performed according to OECD guideline

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the above assessment on oral repeated dose toxicity, Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol does not need to be classified according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.