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Diss Factsheets

Administrative data

Description of key information

The subchronic oral toxicity of 1,2,4-Benzenetricarboxylic acid, decyl octyl ester was investigated in a well-conducted study in Sprague Dawley rats after daily oral administration at dose levels of 50, 200 and 500 mg/kg/day for 13 weeks and recovery from any treatment-related effects during a recovery period of 4 weeks. Signs of treatment-related effects of the test item were observed in males and females dosed at 200 and 500 mg/kg/day. These effects included some changes in clinical pathology parameters and some post mortem findings. The liver was identified as the main target organ on the basis of the organ weight data and histopathological findings. However, these changes were mild and fully reversible. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum. As such, the hepatic effects were not considered to be adverse.
No significant changes were observed in the animals dosed at 50 mg/kg/day. It can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 500 mg/kg/day.



In a subacute 28-day oral toxicity study in rats some treatment-related effects were evident in males and to a minor extent in females at the high dose level of 1000 mg/kg/d.


The adrenals were identified as the main target organs, based on the post-mortem examination.


The observed effects were completely reversible over a 2-week recovery period in the high dose animals. Only mild effects were observed in the animals (essentially males) dosed at 300 mg/kg/d, therefore this dose is expected to be NOAEL.


No effects were observed at the low dose level (NOEL = 100 mg/kg/d).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
31 January 2014 to 30 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with OECD Guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
See details below under 'any other information on materials or methods'
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CD® [Crl:CD®(SD)]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory, Portage, Michigan, USA.
- Age at study initiation: approximately 6 weeks of age
- Weight at study initiation: 30 male and 30 female animals (weighing 234 to 271 g and 178 to 220 g, respectively, at randomization)
- Fasting period before study: No information available.
- Housing: The animals were housed 2 to 3 per cage in solid bottom cages with nonaromatic bedding in an environmentally controlled room. The
animals were individually housed during times of urine collection for clinical pathology analysis. To reduce the potential impact of environmental stressors on the scheduled observations conducted as part of neurobehavioral evaluations, the animals were individually housed in clear, solid bottom cages within the testing environment for at least 12 hours prior to testing. The animals remained individually housed within the testing environment to reduce the potential influence of environmental stressors associated with cage changes and/or movement during the testing period. Animals were re-paired with the appropriate cagemate(s) following completion of these observation periods.
- Diet: Block Lab Diet (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum, except during designated periods. The lot number from each diet lot used for this study was recorded. Certification analysis of each diet lot was performed by the manufacturer.
- Water: Tap water was available ad libitum via an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to SOP.
- Acclimation period: During the 13-day acclimation period, the animals were observed daily with respect to general health and any signs of disease. During the week prior to dose initiation, animals were administered a sham dose of tap water on at least two occasions in the same manner and at the same volume intended for use during the study period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature was continuously monitored, recorded, and maintained to the maximum extent possible within the range of 68 to 79°F.
- Humidity (%): Humidity was continuously monitored, recorded, and maintained to the maximum extent possible within the range of 30 to 70%.
- Air changes (per hr): No information available.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided for approximately 12 hours per day. On occasion, the dark cycle was interrupted due to study activities.

IN-LIFE DATES: From: 13 February 2014 To: 27 March 2014
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: No information provided.
- Concentration in vehicle: 10, 30, and 100 mg/mL.
- Amount of vehicle: dose volume of 10 mL/kg.
- Lot/batch no.: 2CG0041
- Purity: No information available.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples were collected while stirring, using a positive displacement pipette, and placed into amber glass scintillation vials. Samples were stored at room temperature and protected from light until analyzed.

HPLC conditions: Liquid chromatography system equipped with an Agilent Poroshell 120 SB-C8 column, 3.0 x 50 mm, with a gradient flow of 0.1% phosphoric acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.7 mL/minute.

Analysis description: Prior to analysis, duplicate samples were diluted with isopropyl alcohol (diluent) to within the range of the calibration curve. Vehicle samples were diluted with diluent using a dilution factor of 500.000. An aliquot of each sample was injected into the HPLC-UV system for analysis and evaluated at 233 nm.

A total of 40 samples were analyzed for Hatcol® 2942 in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results.

All system suitability tests, performance checks, and calibration curves met the acceptance criteria. Dose formulation samples were analyzed for homogeneity. All test article formulation sample results were within 100% (±5%) average recovery and ≤2.0% RSD.
Therefore, the formulations were considered homogeneous.

Dose formulation samples were analyzed for concentration verification. All samples were within 100±5% average recovery and ≤5% RSD. Test article was not detected in the vehicle control samples. The formulations met the acceptance criteria for study use, and animals received the intended dose.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
0 mg/kg bw/day: 10 per sex (five animals/sex/group were maintained for a 14-day recovery period)
100 mg/kg bw/day: 5 per sex
300 mg/kg bw/day: 5 per sex
1000 mg/kg bw/day: 10 per sex (five animals/sex/group were maintained for a 14-day recovery period)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The regulatory guidelines indicate a limit dose of 1000 mg/kg/day for 90 day studies. Therefore, 1000 mg/kg/day was chosen as the high dose for this study. The mid and low doses are approximate log intervals of the high dose level.
- Rationale for animal assignment: Using a standard, by weight, measured value randomization procedure.
- Rationale for selecting satellite groups: No information provided.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily. On occasion, veterinary consultations were conducted during the course of the study. All treatments and observations were recorded. The medical treatments and observations are not reported but are maintained in the study file.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A cageside clinical examination of each animal was performed daily during the study, except on days of neurobehavioral observations, FOB evaluations, or motor activity measurements. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, and nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior. If warranted, the animal was removed from the cage for closer inspection.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights for all animals were measured and recorded on the day of receipt, prior to randomization, and weekly during the study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: Food consumption was measured and recorded weekly during the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic examinations were conducted on all animals pretest.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to terminal and recovery necropsy. Blood samples (approximately 4.0 mL) were collected via the vena cava after carbon dioxide inhalation.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes. Free access to drinking water but fasted overnight prior to blood collection.
- How many animals: All survivors.
- Parameters checked: leukocyte count (total and absolute differential), erythrocyte count, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration (calculated), absolute reticulocytes, platelet count, blood smear preserve and stain, prothrombin time, activated partial thromboplastin time, fibrinogen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to terminal and recovery necropsy. Blood samples (approximately 4.0 mL) were collected via the vena cava after carbon dioxide inhalation.
- Animals fasted: Yes. Free access to drinking water but fasted overnight prior to blood collection.
- How many animals: All survivors.
- Parameters checked: alkaline phosphatase, total bilirubin (with direct bilirubin if total bilirubin exceeds 1 mg/dL), aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, urea nitrogen, creatinine, total protein, albumin, globulin and A/G (albumin/globulin) ratio (calculated), glucose, total cholesterol, triglycerides, electrolytes (sodium, potassium, chloride), calcium, phosphorus.

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to terminal and recovery necropsy
- Metabolism cages used for collection of urine: Yes. Animals were placed in stainless steel metabolism cages for at least 12 hours to collect urine.
- Animals fasted: No data
- Parameters checked: volume, color and appearance, specific gravity, pH, protein, glucose, bilirubin, ketones, blood, urobilinogen, microscopy of centrifuged sediment.

NEUROBEHAVIOURAL EXAMINATION AND FUNCTIONAL OBSERVATION BATTERY: Yes
- Time schedule for examinations: A neurobehavioral examination of each animal was performed pretest and weekly during the study, except during weeks when FOB evaluations were performed. Observations were made outside the animal’s home cage in a standard arena using an applicable scoring system at approximately the same time (starting within 3 hours of the previous observation) and day, each week. Observations were carefully recorded, and an effort was made to ensure that variations in the test conditions were minimal. FOB evaluations were conducted without knowledge on the part of the testers of the treatment groups on all animals pretest and during Week 4. FOB evaluations included those conducted in the home-cage, during handling, in the open-field, and physiological parameters. During open-field evaluations, each animal was placed in a black plexiglass™ box and observed for a minimum of 3 minutes.
- Dose groups that were examined: All animals.
- Battery of functions tested: The observations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, and reactivity to handling, as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling), difficult or prolonged parturition, or atypical behaviors (e.g., self-mutilation, walking backwards) were also recorded. The parameters evaluated in the FOB were based on those outlined in Moser, et al. The observations included, but were not limited to, evaluation of activity and arousal, posture, rearing, bizarre behavior, clonic and tonic movements, gait, mobility, stereotypy, righting reflex, response to stimulus (approach, click, tail pinch, and touch), palpebral closure, pupil response, piloerection, exophthalmus, lacrimation, salivation, and respiration. Qualitative and/or quantitative measures of defecation and urination were also recorded. Forelimb and hindlimb grip strength was measured using the procedure described by Meyer, et al., and hindlimb splay was quantitatively measured as described by Edwards and Parker. Pain perception was assessed by measuring the latency of response to a nociceptive (thermal) stimulus when each animal was placed on a hot plate apparatus set to 52°C as described by Ankier. Body weight and temperature were also measured.

OTHER:
Locomotor activity evaluations were conducted on all designated animals pretest and 1 to 3 hours postdose during Week 4. Each animal was placed into the assigned Kinder Scientific enclosure for monitoring. The duration of monitoring was 60 minutes with the data summarized into 10-minute segments. A range of different activities were assessed in a three dimensional array and were recorded. Only basic movement, fine movement, rearing, and distance (cm) were used in comparisons between treated and control animals as the most representative activity parameters.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Necropsy examinations were performed under procedures approved by a veterinary pathologist on all animals at the scheduled terminal (day 28) and recovery necropsies (day 42). The animals were euthanized by carbon dioxide inhalation followed by exsanguination via transection of the abdominal vena cava. The animals were examined carefully for external abnormalities including palpable masses. The skin was reflected from a ventral midline incision and any subcutaneous masses were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities. The organs were removed, examined, and, where required, placed in fixative. All designated tissues were fixed in neutral buffered formalin, except for the testes, which were fixed using a modified Davidson’s fixative. Testes were placed into formalin following fixation. Formalin was infused into the lung via the trachea and into the urinary bladder.
A full complement of tissues and organs was collected from all animals:
- Adrenal (2)
- Bone with marrow [femur]
- Bone with marrow [sternum]
- Bone marrow smear [2 collected]
- Brain [cerebrum, midbrain, cerebellum, medulla/pons, olfactory bulbs]*
- Epididymis (2)*
- Gastrointestinal tract: esophagus, stomach [glandular and nonglandular], duodenum, jejunum, ileum, cecum, colon, rectum
- Gonads: ovary (2) with oviduct (2), testis (2)*
- Gross lesions
- Heart*
- Kidney (2)*
- Liver [3 sections collected; 2 examined]*
- Lung with bronchi [collected whole; 2 sections examined]
- Lymph nodes: mandibular [2 collected; 1 examined] and mesenteric
- Nerve, peripheral [tibial]
- Peyer’s patch
- Prostate and seminal vesicle (2)
- Sciatic nerve
- Spinal cord [cervical, thoracic, and lumbar]
- Spleen*
- Thymus*
- Thyroid/parathyroid (2)
- Tongue
- Trachea
- Urinary bladder
- Uterus [both horns]/Cervix
- Vagina

(2):Paired organ
*: Weighed organ

Organ Weights
Body weights and protocol-designated organ weights were recorded for all animals at the scheduled necropsies and appropriate organ weight ratios were calculated (relative to body and brain weights). Paired organs were weighed together.

HISTOPATHOLOGY: Yes
Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The adrenal gland, liver, and mesenteric lymph node were determined to be potential target organs and were examined for all dose groups. The slides were examined by a board-certified veterinary pathologist. A four-step grading system was utilized to define gradable lesions for comparison between dose
groups.
Statistics:
The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group. Data for some endpoints were transformed by either a log or rank transformation prior to conducting the specified analysis.

Types of Analyses conducted: Group Pair-wise Comparisons (Levene’s/ANOVADunnett’s/Welch’s), Log Transformation/Group Pair-wise Comparisons, Rank Transformation with Dunnett’s Test, Cochran Mantel Haenszel Test, Analysis of Variance with Adjustment for Multiple Comparisons.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males given 1000 mg/kg/day and females given 100 or 300 mg/kg/day had lower body weights and decreased body weight gain. All were considered test article-related. However, none of these findings were considered adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males given 1000 mg/kg/day and females given 100 or 300 mg/kg/day had decreased food consumption. All were considered test article-related. However, none of these findings were considered adverse.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Mild increases in total leukocytes, lymphocytes and neutrophils and mild decreases in fibrinogen. Test article related but not considered adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mild to moderate increases in ALP, AST, and ALT, mild decreases in globulin and albumin and total protein and mild increases in serum lipids. Test article related but not considered adverse.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Increased urine volume and decreased urine specific gravity in both sexes administered 1000 mg/kg/day. Test article related but not considered adverse.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test article-related alterations in mean organ weights were observed in the thymus, spleen, liver, and adrenal glands but not considered adverse.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test article-related microscopic findings were observed in the liver, adrenal glands and mesenteric lymph nodes but not considered adverse.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived to their scheduled sacrifice.
No test article-related findings were reported. One male given 1000 mg/kg/day (Animal number 447) had sparse hair, and one female given 1000 mg/kg/day (Animal number 454) had a portion of the tail missing. However, these signs were in just these animals,were considered incidental findings, and were not considered test article-related.

BODY WEIGHT AND WEIGHT GAIN
Lower body weight and decreased body weight gain were observed in the males given Hatcol® 2942 and in the females given 100 or 300 mg/kg/day after 4 weeks of dosing. Body weights in males given 100 or 300 mg/kg/day were less than 7% lower than the average control males, but males given 1000 mg/kg/day on average weighed 12.8% less than the average male control body weight and gained 32.5% less weight over 4 weeks of dosing than the control males. This same change was not observed in females given 1000 mg/kg/day, but a 9 to 10% body weight decrease was observed in the females given 100 or 300 mg/kg/day, and a 34.8% or 26.2% decrease in body weight gain was observed in the females given 100 or 300 mg/kg/day respectively. By the end of the recovery period, a 6.4 or 11.0% decrease in the body weight of the females and males given 1000 mg/kg/day, respectively, was observed. While the males given 1000 mg/kg/day gained about as much as the control males during the recovery period, females given 1000 mg/kg/day did not. Lower body weights and decreased body weight gain were considered test article-related. However, since there were no clinical observations noted in this study, the body weight changes were not considered adverse.

FOOD CONSUMPTION
Weekly values were not significantly different between groups, but over 4 weeks, the total food consumption in males given 1000 mg/kg/day was 19.9% less than the control males. Females given 300 mg/kg/day ate 17.4% less than the control females during the 4 week dosing period. These food consumption differences were considered test article-related and correlated with the lower body weights and decreased body weight gain also noted in these groups. Since there were no clinical observations noted in this study, the food consumption changes were not considered adverse. Food consumption for the males and females in the other groups given Hatcol® 2942 differed from the control animals by less than 8% and was not considered physiologically important or test article-related.

OPHTHALMOSCOPIC EXAMINATION
No abnormalities were detected at the pre-test ophthalmic examination.

HAEMATOLOGY
At the terminal collection, males and females administered 1000 mg/kg/day had mild increases in total leukocytes (up to +66%), which were attributable to mild increases in lymphocytes (up to +68%) and neutrophils (up to +57%), relative to controls. These leukocyte effects were considered test article-related, but lacked a microscopic correlate, and were fully resolved at the recovery collection. All other fluctuations among hematology endpoints were within expected ranges for biologic and procedure-related variation.

Coagulation: At the terminal collection, males administered ≥300 mg/kg/day and females administered 1000 mg/kg/day had mild statistically significant decreases in fibrinogen (up to -39%), relative to controls. Decreases in fibrinogen were considered test article-related, correlated with erythrophagocytosis in the mesenteric lymph node, and were fully resolved at the recovery collection. APTT was also mildly statistically decreased (-12%) in females administered 1000 mg/kg/day, relative to controls. This decrease in APTT was potentially test article-related, but was not present in males, and was considered resolved at the recovery collection.

CLINICAL CHEMISTRY
At the terminal collection, there was evidence of hepatobiliary and hepatocellular injury in males and females administered 1000 mg/kg/day, as illustrated by mild to moderate statistically significant increases in alkaline phosphatase (ALP; up to +261%), aspartate aminotransferase (AST; up to +39%) and alanine aminotransferase (ALT; up to +203%), relative to controls. These effects were considered test article-related, and correlated with hepatocellular hypertrophy as further described in the histopathology report. Increases in enzyme activity were generally resolved at the recovery collection. At the terminal collection in males and females administered 1000 mg/kg/day, there were mild statistically significant decreases in total protein (up to -17%), which were attributable to mild to moderate decreases in globulin (up to -25%) and albumin (up to -10%), relative to controls. These effects resulted in mildly increased albumin/globulin ratios (up to +20%), and were considered test article-related. Effects on serum proteins were likely associated with erythrophagocytosis in the mesenteric lymph node, as further described in the histopathology report. Reductions in total protein, globulin and albumin were considered resolved at the recovery collection.

Mild effects on serum lipids were also noted at the terminal collection in males and females administered 1000 mg/kg/day, including mild increases in triglyceride (up to +77%) in both sexes, as well as a mild increase in cholesterol (68%) in males only. These effects may indicate alterations in lipid hemostasis, and were considered test article-related. They were resolved at the recovery collection. All other fluctuations among clinical chemistry endpoints were within an expected range for biologic and procedure-related variation.

URINALYSIS
At the terminal collection, males and females administered 1000 mg/kg/day had mild to moderate increases in urine volume (up to +467%) with corresponding reductions in urine specific gravity, relative to controls. These changes indicated increased urine production and/or decreased urine concentration, but lacked clinical or microscopic correlates. Effects on urine volume and specific gravity were considered test article-related and fully resolved at the recovery collection. No other test article-related alterations were observed among urinalysis parameters in any treatment group at the terminal or recovery collections. There were minor variations between treatment groups among physical (appearance) and biochemical (protein, occult blood, etc.) urinary components; however, all findings were considered within expected ranges for biologic and/or procedure-related variability.

NEUROBEHAVIOUR AND FUNCTIONAL OBSERVATION BATTERY
There were no differences between the control and treated animals that were considered test article-related. The thermal response in males given 300 mg/kg/day was increased on Week 4 when compared to the control males, and the increase was a result of measurements that were much higher than the pretest values in three of the five males tested in this group. Since there were no other findings that could be associated with an increased thermal response, and it was not noted in any other group of animals given Hatcol® 2942, the increased thermal response in males given 300 mg/kg/day was not considered test article related.

ORGAN WEIGHTS
At the terminal necropsy, test article-related alterations in mean organ weights were observed in the thymus, spleen, liver and adrenal glands (see following table). Increased liver weight correlated to hepatocellular hypertrophy observed in males given 300 or 1000 mg/kg/day; no microscopic correlation was observed in females. Adrenal gland hypertrophy was observed by sub-gross examination of the slides in males given 1000 mg/kg/day and in females given 300 or 1000 mg/kg/day.
At the recovery necropsy, test article-related alterations in organ weights were observed in the thymus, spleen and adrenal glands of males and females and the liver of males.

GROSS PATHOLOGY
No test article-related macroscopic findings were observed.

HISTOPATHOLOGY:
For the terminal necropsy, test article-related microscopic findings were observed in the liver, adrenal glands and mesenteric lymph nodes. In the liver, minimal hepatocellular hypertrophy was observed in 2/5 and 3/5 males given 300 or 1000 mg/kg/day, respectively and in 1/5 females given 1000 mg/kg/day. Increased liver weight and hepatocyte hypertrophy observed in some animals given 1000 mg/kg/day were associated with an increase in liver enzymes [Total Alkaline Phosphatase (ALP), Aspartate Aminotransferase (AST), and Alanine Aminotransferase (ALT)] noted in clinical chemistry evaluation. Adrenal gland enlargement compared with controls was observed by sub-gross examination of the slides and corresponded to minimal diffuse cortical hypertrophy of the zona fascicularis in 3/5 males given 1000 mg/kg/day, 1/5 females given 300 mg/kg/day, and 5/5 females given 1000 mg/kg/day (because of variable planes of section, adrenal gland weight and sub-gross examination of the slides are considered more accurate evaluations than microscopic exam alone for cortical hypertrophy). Minimal to mild erythrophagocytosis was observed in the medullary sinus of the mesenteric lymph node in 1/5, 3/5 and 4/5 males given 100, 300 or 1000 mg/kg/day, respectively, in one control female and in 5/5 females given 1000 mg/kg/day. Erythrophagocytosis in the mesenteric lymph node is a commonly observed background finding; however, it was observed with increased incidence in test article-treated animals compared with concurrent controls and there was a correlation with decreased proteins noted on clinical chemistry evaluation in animals given 1000 mg/kg/day. There were no microscopic correlations to the increased spleen weights or decreased thymus
weights. For the recovery necropsy, target tissues identified during the terminal phase were examined. Adrenal gland enlargement compared with controls was observed by sub-gross examination of the slides and corresponded to minimal diffuse cortical hypertrophy of the zona fascicularis in 2/5 females given 1000 mg/kg/day. All other findings were considered incidental or background.

OTHER FINDINGS
LOCOMOTOR ACTIVITY
No test article related-changes were observed. Rearing count was increased in males given 100 mg/kg/day. Since it was not increased at any higher dose level given to males or in females given Hatcol® 2942, the increase rearing count was not considered test article-related.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects compared with controls at 1000 mg/kg bw/day (highest dose tested).
Critical effects observed:
not specified
Conclusions:
In conclusion, administration of Hatcol® 2942 was well tolerated in CD® [Crl:CD®(SD)] rats as a once daily oral gavage for 28 days at dose levels of 0, 100, 300, and 1000 mg/kg/day. Males given 1000 mg/kg/day and females given 100 or 300 mg/kg/day had lower body weights, decreased body weight gain, and/or decreased food consumption. Increases in ALT, AST, and ALP were noted in animals given 1000 mg/kg/day which correlated with hepatocellular hypertrophy. In addition, adrenal gland enlargement in animals given 1000 mg/kg/day and erythrophagocytosis in the medullary sinus of the mesenteric lymph node in all treated groups was observed, and all were considered test article-related. However, none of these findings were considered adverse. Therefore, the no-observed adverse- effect-level (NOAEL) in rats given Hatcol® 2942 daily for 28 days is 1000 mg/kg/day.
Executive summary:

This study was conducted for Chemtura Corporation to evaluate the potential toxicity of the test article (Hatcol® 2942) after oral gavage administration for at least 28 days and to evaluate reversibility, progression, or delayed appearance of any observed changes following a 14-day postdose observation period.

Assessment of toxicity was based on mortality, clinical observations, neurobehavioral observations, body weight, and food consumption; functional observational battery (FOB) evaluations; monitoring of locomotor activity; and anatomic and clinical pathology. Ophthalmoscopic examinations were conducted pretest.

All animals survived to their scheduled sacrifice. No test article-related clinical findings were reported, and no neuro-behavioral or locomotor differences were observed.

Lower body weight and decreased body weight gain was observed in the males given Hatcol® 2942 and in the females given 100 or 300 mg/kg/day after 4 weeks of dosing. By the end of the recovery period, a 6.4 or 11.0% decrease in the body weight of the females and males given 1000 mg/kg/day, respectively, was observed. Lower body weights and decreased body weight gain were considered test article-related, but not adverse. Over the four weeks of dosing, the total food consumption in males given 1000 mg/kg/day and females given 300 mg/kg/day was less than that consumed by the control animals. Decreased food consumption was considered test article-related and correlated with the lower body weights and decreased body weight gain also noted in these groups, but the food consumption changes were not considered adverse.

Once daily oral administration of Hatcol® 2942 at doses of 100, 300, or 1000 mg/kg/day for up to 28 consecutive days to rats resulted in mild to moderate increases in ALP, AST, and ALT, mild decreases in in fibrinogen, globulin and albumin and total protein , mild increases in total leukocytes, lymphocytes and neutrophils, and mild increases in serum lipids. These changes were often noted in both sexes administered 1000 mg/kg/day, but less evident or not noticed at 100 or 300 mg/kg/day. These effects were generally resolved at the recovery collection.

No test article-related macroscopic findings were observed. Test article-related alterations in mean organ weights were observed in the thymus, spleen, liver, and adrenal glands. Increased liver weight correlated to hepatocellular hypertrophy observed in males given 300 or 1000 mg/kg/day. Adrenal gland hypertrophy was observed by sub-gross examination of the slides in males given 1000 mg/kg/day and in females given 300 or 1000 mg/kg/day. Test article-related microscopic findings were observed in the liver, adrenal glands and mesenteric lymph nodes. In the liver, minimal hepatocellular hypertrophy was observed in males given 300 or 1000 mg/kg/day, and in one female given 1000 mg/kg/day. Adrenal gland enlargement compared with controls was observed by sub-gross examination of the slides and corresponded to minimal diffuse cortical hypertrophy of the zona fascicularis in males and females given 1000 mg/kg/day, and in females given 300 mg/kg/day. Minimal to mild erythrophagocytosis was observed in the medullary sinus of the mesenteric lymph node in males given 100, 300, or 1000 mg/kg/day, in one control female and in all females given 1000 mg/kg/day. Erythrophagocytosis in the mesenteric lymph node was observed with increased incidence in test article-treated animals compared with concurrent controls, and there was a correlation with decreased proteins noted on clinical chemistry evaluation in animals given 1000 mg/kg/day. There were no microscopic correlations to the increased spleen weights or decreased thymus weights.

In conclusion, administration of Hatcol® 2942 was well tolerated in CD® [Crl:CD®(SD)] rats as a once daily oral gavage for 28 days at dose levels of 0, 100, 300, and 1000 mg/kg/day. Males given 1000 mg/kg/day and females given 100 or 300 mg/kg/day had lower body weights, decreased body weight gain, and/or decreased food consumption. Increases in ALT, AST, and ALP were noted in animals given 1000 mg/kg/day which correlated with hepatocellular hypertrophy. In addition, adrenal gland enlargement in animals given 1000 mg/kg/day and erythrophagocytosis in the medullary sinus of the mesenteric lymph node in all treated groups was observed, and all were considered test article-related. However, none of these findings were considered adverse. Therefore, the no-observedadverse-effect-level (NOAEL) in rats given Hatcol® 2942 daily for 28 days is 1000 mg/kg/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
June to July 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented study accordingt o OECD Guideline and GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted October 3, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l.
- Age at study initiation: 27-29 days
- Weight at study initiation: 88-107 g
- Fasting period before study: no
- Housing: 5 of one sex in clear polycarbonate cages with a stainless steel mesh lid and floor
- Diet: rodent diet (4 RF 21, Mucedola S. r. l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) ad libitum
- Water: drinking water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15-25
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: June 4 2009 To: July 16, 2009
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 20, 60, and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were diluted with 2-propanol and an internal standard was added. The dilutions were then analysed by GC/FID.

Analysis of samples of the formulations prepared on day 1 and week 4 to check the homogeneity and concentration
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5

additional 5 animals per sex in high dose and control group as satellite groups for recovery assessment
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 2 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and once daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and once a week thereafter
BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption by each cage of rats was recorded at weekly intervals and mean daily diet consumption calculated as g food/animal/day.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of week 4 of treatment and at the end of week 2 of the recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells), platelets, prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of week 4 of treatment and at the end of week 2 of the recovery period
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, urea, creatinine, glucose, triglycerides, bile acids, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chloride


URINALYSIS: Yes
- Time schedule for collection of urine: at the end of week 4 of treatment and at the end of week 2 of the recovery period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: appearance, volume, specific gravity, pH, protein, total reducing substances, glucose, ketones, bilirubin, urobilinogen, blood; epithelial cells, leucocytes, erythrocytes, chrystals, spermatozoa and precursors, other abnormal components


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before commencement of treatment and once a week thereafter
- Dose groups that were examined: all

OTHER:
- motor activity assessment, sensory reactivity to stimuli of different modalities and assessment of grip strength (once during week 4 of treatment and once during week 2 of recovery)
- hormone determination (T3, T4 and THS)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, post mortem examination including examination of the external surface and orifices; determination of organ weights (see table 1)

HISTOPATHOLOGY: Yes (see table 2)

Histopathological evaluation was performed on all males and females of the control group and of the high dose group (receiving the test item at 1000 mg/kg/day), sacrificed at the end of treatment. All abnormalities from all main phase animals were also examined.
Particular attention was paid to the gonads (ovaries and testes), accessory sex organs (uterus including cervix, epididymides, seminal vesicles with coagulation glands, dorsolateral and ventral prostate), vagina, pituitary, thyroid and adrenal glands for the evaluation of possible endocrine-related effects. In addition, as indicated by the international test program, synchronization of the oestrus cycle was also evaluated to better identify eventual disturbances on female sex hormone homeostasis.
The histopathological evaluation was performed following the suggestions provided by the OECD Test Guideline 407 (which refers to "Endocrine sisruption: A guidance document for histologic evaluation of endocrin and reproductive tests", Draft 3, May 16 2008).
On the basis of the pathological changes noted in the adrenals of high dose males and in the stomach of a few animals in the same treated group, the examination was extended to include the stomach, from males and females receiving the test item at dosages of 100 and 300 mg/kg/day, and the adrenals from the males dosed at 100 and 300 mg/kg and in the males killed after the recovery period.
Statistics:
For continued variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treatment group and the control group were assessed by Dunnet's test. The homogeneity of the data was verified by Bartlett's test before Dunnet's test.
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study. Clinical signs were limited to hair loss, observed in a total of 6/10 females dosed at 1000 mg/kg/day from Day 8 of treatment up to the end of the recovery period.

BODY WEIGHT AND WEIGHT GAIN (see table 3)
A trend towards a decrease in mean body weight, compared to controls, (from 5% to 9%) was recorded in the males receiving 1000 mg/kg/day from Day 8 throughout the administration period, up to the end of the recovery period. No changes of note were seen in the other male groups and in treated females. The body weight reductions noted in all animals on Day 29, when compared to Day 22, were due to the overnight fast preceding the bleeding procedures for clinical pathology analyses.

FOOD CONSUMPTION
Slight reductions in food intake were observed in the males dosed at 1000 mg/kg/day from Day 8 (-17%) to Day 29 (-7%). No changes were seen in the other male groups and in treated females. No differences between treated animals and controls were observed at the end of recovery.
The above changes were not considered to be toxicologically relevant as they were slight and comparable to the historical control data.
The food consumption reductions, noted in all animals on Day 29 when compared to Day 22, were due to the overnight fast preceding the bleeding procedures for clinical pathology analyses.

HAEMATOLOGY (see table 4a and 4b)
Leucocytosis was recorded in animals treated with 1000 mg/kg/day and in females dosed with 300 mg/kg/day. The increase in white blood cells mainly involved the lymphocytes and was 25% to 60% above controls.
The other statistically significant changes recorded, such as an increase in erythrocytes, haemoglobin and haematocrit, observed in males treated with 300 and/or 1000 mg/kg/day, a decrease in reticulocytes, seen in those receiving 1000 mg/kg/day, and of prothrombin time seen in males treated with 100 mg/kg/day and in females dosed with 1000 mg/kg/day, were of low magnitude and inconsistent between sexes, therefore they were considered to be of no toxicological significance. In the recovery group leucocytosis was still present in females treated with 1000 mg/kg/day (increased by 27% compared with controls.

CLINICAL CHEMISTRY (see table 5)
A moderate to severe increase in alkaline phosphatase (+67% and 6-fold higher for males of the mid- and high dose groups, +5-fold for high-dose females) and bile acids (+9 and 3-fold in high dose males and females, respectively) and a decrease in globulin were observed in males treated with 300 and 1000 mg/kg/day and in females dosed with 1000 mg/kg/day. In addition, increments of alanine aminotransferase (2-fold), gamma-glutamyl transferase (3-fold) and decrements of bilirubin (-27%), protein and sodium were observed in males dosed with 1000 mg/kg/day.
Females from the same groups showed additional increments of alanine aminotransferase (2.8-fold), aspartate aminotransferase (+36%), gamma-glutamyltransferase (+29%) and urea (+33%) and decrements of bilirubin (-40%), protein, sodium and calcium. Some of the above mentioned changes could be related to the modifications seen in the adrenals recorded at the histopathological examination and to the related increase in corticosteroids. The other statistically significant changes were considered to be incidental as they were not dose-related and/or inconsistent between sexes .

Changes recorded during the dosing phase showed a complete reversibility in the recovery groups. Even though phosphorus, in males dosed with 1000 mg/kg/day, and urea, in females from the same group, did not decrease, their mean values were comparable with controls.
The statistically significant changes detected (triglycerides, chloride and sodium in males, aspartate aminotransferase in females) were not seen during the dosing phase, therefore they were considered to be incidental.

URINALYSIS
No changes of toxicological significance were recorded. The differences of specific gravity between control and treated females were of low severity. In the recovery phase no changes were observed.

NEUROBEHAVIOUR
No treatment-related changes were found at the weekly neurotoxicity evaluation.

ORGAN WEIGHTS (see table 6)
Slight but statistically significant increases in absolute (+16% and 33%) and relative (+29% and 34%) liver weights were recorded at the end of the treatment period in both males and females of the highest dose group in comparison with the respective controls. In addition, the absolute and relative adrenal weights were increased in the males dosed at 1000 mg/kg/day (+8% and 20%, respectively). These changes were found to be completely recovered after 2 treatment-free weeks. The toxicological significance of the above changes is supported by the correlation with the clinical and microscopic pathology findings. No other changes of toxicological significance were reported.

GROSS PATHOLOGY
The only relevant change noted in treated animals, when compared with controls, during the post mortem examination, was a minimal (on one occasion moderate) hair loss on the skin of the head, detected in 4/5 females dosed at 1000 mg/kg/day. In the recovery group hair loss was again noted on the forelimbs and head region of females dosed at 1000 mg/kg/day and sacrificed after the recovery period. The remaining changes observed as enlarged ovaries, distended with clear fluid content in the uterus, swollen spleen or red colour of the thymus are considered to be incidental in origin and characteristically seen in untreated Sprague Dawley rats of the same age, under our experimental conditions.

HISTOPATHOLOGY:

Treatment-related changes were seen in the adrenals of the high dose males. This change consisted of cortical cell hypertrophy associated with vacuolation, as fatty change, primarily involving the zona fascicolata.
No similar changes were noted in the males receiving the test item at 100 and 300 mg/kg/day, sacrificed at the end of the treatment period or in those sacrificed at the end of the recovery period.
The evaluation of the reproductive system of male (testis, epididymis, prostate, coagulating gland and seminal vesicles) and female (ovary, uterus and vagina) animals did not show any pathological alteration in the spermatogenesis as well no irregularity in the oestrus cycle.

Squamous hyperplasia of the forestomach mucosa associated with gastric inflammation was only detected in one male and one female, dosed at 1000 mg/kg/day. Males and females dosed at 100 and 300 mg/kg/day did not show any alteration in the stomach.
This sporadic pathological change could be considered incidental and probably related to an individual stress-related change.
The remaining lesions reported were considered as an expression of spontaneous pathology or as incidental findings, commonly observed in this species at this age and under our experimental conditions.


OTHER FINDINGS
No significant differences between treated animals and controls were observed at the evaluation of sensory reaction performed at the end of treatment. Motor activity measurements performed at the end of the treatment period did not show changes which could be ascribed to treatment.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Critical effects observed:
not specified
Table 3: Body weights of males (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d
day 1 212.17 ± 8.62 207.60 ± 7.77 201.87 ± 6.58 206.30 ± 7.82
day 8 261.78 ± 12.72 254.79 ± 8.80 247.26 ± 9.25 247.53 ± 110.39*
day 15 309.27 ± 15.05 298.20 ± 11.60 288.67 ± 12.22* 284.52 ± 11.81**
day 22 339.44 ± 17.19 327.37 ± 9.40 320.53 ± 16.65 311.11 ± 15.12**
day 29 340.78 ± 19.25 329.45 ± 12.26 317.14 ± 19.98 309.66 ± 10.99**
Recovery phase
Day 1 365.39 ± 21.24 326.39 ± 17.49*
Day 8 382.19 ± 23.58 340.30 ± 19.49*
Day 15 377.09 ± 24.86 337.03 ± 22.06*
*p < 0.05; ** p < 0.01
Table 4a: Haematology findings in males (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d
Red blood cells (*106/µl) 7.316 ± 0.133 7.574 ± 0.248 7.635 ± 0.162 7.798 ± 0.258**
Haemaglobin (g/dl) 14.06 ± 0.29 14.44 ± 0.44 14.83 ± 0.43* 15.04 ± 0.29**
Haematocrit (%) 40.24 ± 0.62 41.30 ± 1.42 42.25 ± 1.08* 43.44 ± 1.06**
Reticulocytes 137.04 ± 26.46 123.04 ± 6.83 1325.25 ± 18.78 104.14 ± 12.13*
White cell blood count (*103/µl) 11.148 ± 2.220 9.3486 ± 0.805 10.458 ± 2.149 13.282 ± 1.959
Lymphocytes (*103/µl) 8.712 ± 2.307 7.332 ± 0.880 8.425 ± 1.941 10.866 ± 2.217
*p < 0.05; ** p < 0.01
Table 4b: Haematology findings in females (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d Recovery control group Recovery group 1000 mg/kg/d
White cell blood count (*103/µl) 6.208 ± 1.028 5.684 ± 1.362 9.174 ± 1.010** 9.930 ± 1.261** 7.222 ± 0.704 9.198 ± 1.196
Lymphocytes (*103/µl) 5.196 ± 0.756 4.752 ± 1.196 7.838 ± 0.838** 8.410 ± 1.090** 6.016 ± 0.616 7.808 ± 1.190*
*p < 0.05; ** p < 0.01
Table 5: Clinical chemistry findings (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d Recovery control group Recovery group 1000 mg/kg/d
Alkaline phosphatase (U/l) males 357.96 ± 28.25 398.90 ± 97.75 597.06 ± 106.54** 2212.06 ± 429.06** 276.30 ± 29.77 301.44 ± 15.90
females 287.54 ± 26.85 336.94 ± 93.45 361.86 ± 61.66 1421.66 ± 631.98* 184.30 ± 20.87 189.12 ± 26.00
Alanine Amino-transferase (U/l) males  51.52 ± 17.46 35.70 ± 5.23 43.38 ± 5.97 103.24 ± 17.68** 40.56 ± 2.93 41.74 ± 5.05
females 30.68 ± 12.08 26.42 ± 3.94 25.32 ± 4.21 86.06 ± 26.53* 31.40 ± 1.57 30.66 ± 3.45
Aspartate Amino-transferase (U/l) females 57.70 ± 6.29 57.26 ± 4.39 60.26 ± 4.94 78.40 ± 4.99** 84.10 ± 5.43 73.94 ± 3.59**
¿-Glutamyl transferase (U/l) males 1.34 ± 1.02 0.88 ± 0.28 1.98 ± 0.56 4.12 ± 1.45* 2.08 ± 2.00 2.56 ± 1.03
females 0.70 ± 0.23 0.78 ± 0.13 0.60 ± 0.19 0.90 ± 0.16 2.50 ± 1.31 1.86 ± 1.02
Bile acids (µmol/l) males 10.08 ± 5.72 9.10 ± 5.25 16.12 ± 3.88 89.96 ± 29.39** 20.46 ± 11.16 8.54 ± 5.26
females 15.46 ± 8.78 16.14 ± 10.07 5.06 ± 2.75 43042 ± 32.32 9.44 ± 8.02 7.68 ± 3.80
Bilirubin (mg/dl) males 0.188 ± 0.018 0.192 ± 0.033 0.188 ± 0.026 0.138 ± 0.026* 0.160 ± 0.034 0.170± 0.035
females 0.226 ± 0.024 0.186 ± 0.026 0.232 ± 0.022 0.136 ± 0.027** 0.216 ± 0.038 0.230 ± 0.014
Protein (g/dl) males 5.96 ± 0.05 5.90 ± 0.14 5.88 ± 0.30 5.24 ± 0.18** 6.48 ± 0.26 6.60 ± 0.55
females 5.42 ± 0.11 5.42 ± 0.23 5.58 ± 0.27 4.78 ± 0.26** 6.34 ± 0.15 6.24 ± 0.11
Globulin males 2.30 ± 0.12 2.20 ± 0.07 2.08 ± 0.16* 1.64 ± 0.09** 2.38 ± 0.52 2.66 ± 0.53
females 1.94 ± 0.15 1.92 ± 0.16 1.96 ± 0.15 1.50 ± 0.14** 2.32 ± 0.08 2.38 ± 0.08
Albumin/Globulin ratio males 1.60 ± 0.13 1.68 ± 0.08 1.83 ± 0.14* 2.20 ± 0.12** 1.82 ± 0.57 1.53 ± 0.30
females 1.80 ± 0.18 1.83 ± 0.15 1.85 ± 0.13 2.20 ± 0.16** 1.74 ± 0.11 1.62 ± 0.09
Urea (mg/dl) females 37.54 ± 5.56 43.48 ± 3.56 39.34 ± 5.30 49.88 ± 3.81** 66.14 ± 16.19 66.00 ± 1.97
Sodium (mmol/l) males 149.76 ± 1.20 152.00 ± 1.12 149.50 ± 1.34 145.92 ± 1.76** 148.22 ± 1.53 151.22 ± 1.05**
females 150.28 ± 1.64 150.62 ± 1.68 148.92 ± 2.27 146.62 ± 0.52** 150.16 ± 1.25 148.18 ± 1.54
Calcium (mmol/l) females 2.322 ± 0.077 2.310 ± 0.022 2.342 ± 0.047 2.200 ± 0.063** 2.582 ± 0.058 2.578 ± 0.047
*p < 0.05; ** p < 0.01
Table 6: Organ weights (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d Recovery control group Recovery group 1000 mg/kg/d
Liver (absolute) males 10.279 ± 1.304 9.713 ± 0.500 9.874 ± 0.957 11.960 ± 1.125* 9.981 ± 0.826 9.013 ± 0.997
females 6.201 ± 0.371 6.646 ± 0.693 6.395 ± 0.413 8.229 ± 0.401** 5.907 ± 0.526 6.299 ± 0.430
Liver (relative) males 3.009 ± 0.217 2.954 ± 0.106 3.118 ± 0.165 3.868 ± 0.246** 2.671 ± 0.103 2.688 ± 0.135
females 2.765 ± 0.080 2.960 ± 0.201 2.934 ± 0.052 3.697 ± 0.194** 2.627 ± 0.200 2.727 ± 0.100
Adrenal (absolute) males 0.0536 ± 0.0080 0.0510 ± 0.0022 0.0480 ± 0.0031 0.0580 ± 0.0056 0.0552 ± 0.0053 0.0506 ± 0.0059
females 0.0674 ± 0.0084 0.0664 ± 0.0060 0.0656 ± 0.0051 0.0662 ± 0.0056 0.0584 ± 0.0047 0.0688 ± 0.0066*
Adrenal (relative) males 0.0157 ± 0.0022 0.0155 ± 0.0005 0.0152 ± 0.0016 0.0188 ± 0.0013* 0.0148 ± 0.0018 0.0152 ± 0.0019
females 0.0301 ± 0.0038 0.0297 ± 0.0027 0.0301 ± 0.0024 0.0297 ± 0.0018 0.0261 ± 0.0028 0.0298 ± 0.0019*
*p < 0.05; ** < 0.01
Conclusions:
On the basis of the results, some treatment-related effects were evident in males and, to a minor extent in females at the high dose level of 1000 mg/kg/d. The adrenals were identified as the main target organs, based on the post-mortem examination. The observed effects were completely reversible over a 2-week recovery period in the high dose animals. Only mild effects were observed in the animals (essentially males) dosed at 300 mg/kg/d. No effects were observed at the low dose level (100 mg/kg/d).
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
04-NOV-2021 to 18-NOV-2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Preliminary dose range-finding study designed to allow selection of appropriate dose levels for a subsequent OECD Guideline 421 study.

GLP compliance:
no
Remarks:
Preliminary dose range-finding study
Limit test:
yes
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for toxicology studies. The Wistar rat is one of the standard strains used for repeat-dose toxicity studies including reproductive and developmental toxicity studies. Adequate historical database is available at the Test Facility for this strain of rat.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany).
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation: Young adult rats, 8 weeks old.
- Weight at study initiation: 312-351 g males, 203-224 g females
- Fasting period before study: no
- Housing: Rodents were group housed (2 animals/sex/group per cage) in Type II and/or III polycarbonate cages.
- Diet : Ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.
- Water: tap water from the municipal supply, as for human consumption, from a 500 mL bottle, ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The standard content of the diet as provided by the Supplier and a copy of the Certificate of Analysis (batch number: 536 84829, expiry date: 31 May 2022) was archived with the raw data at Charles River Laboratories Hungary Kft. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of
State Public Health and Medical Officer Service. The quality control results are included in the raw data and archived at Charles River Laboratories Hungary Kft. The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1-22.4°C ( Target range: 19 - 25°C)
- Humidity (%): 28-60% (Target range: 30 - 70%)
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 04-11-2021 To: 18-11-2021
Route of administration:
oral: feed
Details on route of administration:
Oral administration was considered the most appropriate route of administration based on potential human exposure.
Vehicle:
other: ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice- Breeding and Maintenance
Details on oral exposure:
PREPARATION OF DIETARY FORMULATIONS:
The test item was incorporated into the diet at dose concentrations of 1300, 4500 and 13000 ppm by mixing. Water was used for dampening the diet for pelleting. Following mixing, pellets were prepared by simple compression; A similar diet preparation procedures were used to generate control diet (0 mg test item /kg diet).The pelleting process was considered not to induce an "unmixing" of the diets or particle segregation and would prevent the potential settling out of the fine-heavy particles that could occur in handling/transport of powder diets.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was selected to be administered in the diet as it is one of the possible routes of human exposure.
- Concentration in vehicle: 0, 1300, 4500, 13000 ppm
- Amount of vehicle (if gavage): NA
- Lot/batch no. (if required): Batch number: 536 84829
- Purity: Not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of concentration and/or homogeneity in the dietary formulations was performed using extraction from diet and a validated GC-FID method. Test item-containing diet samples of an appropriate weight were collected from the diet of each dose group before the start of treatment with the batch.
Representative dietary formulation samples (at least 5 g/sample) were collected from five different places of the diet container from each dose group, additionally one sample from the middle of the diet container were collected for the control diet.


Duration of treatment / exposure:
14 consecutive days
Frequency of treatment:
Daily, a constant concentration of test item in diet was administered daily to all animals.
Dose / conc.:
0 ppm
Remarks:
Group 1- Control
Dose / conc.:
1 300 ppm
Remarks:
Group 2 - Low dose
Dose / conc.:
4 500 ppm
Remarks:
Group 3 - Mid dose
Dose / conc.:
13 000 ppm
Remarks:
Group 4 - High dose
No. of animals per sex per dose:
6/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The oral route was selected as it is one of the possible routes of human exposure and will be the route used in the upcoming OECD 421 study for which this DRF study provided data to inform the selection of dose levels. The test item was administered in the diet for palatability assessment. The concentrations of the test item in ssniff® SM R/M-Z+H “Complete Diet for Rats and Mice- Breeding and Maintenance” were selected by the Sponsor in consultation with the Study Director, based on the available data and information.

- Rationale for animal assignment:
All animals were weighed on the day before the start of the treatment period and randomly allocated to study groups. The results of the randomization were checked using a computer program to verify the homogeneity and deviations between the groups. Males and females were randomised separately.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day), general cage side observations were made daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena prior to the first treatment (to allow for within-subject comparisons) and daily thereafter.
Any clinical signs, pertinent behavioral changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Any signs of neurotoxicity were specifically documented and reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomisation then daily for 4 days then twice weekly and prior to necropsy on Day 14.

FOOD CONSUMPTION: Yes
- The determination of food consumption was performed for all groups on Day 0, then daily for 4 days then twice weekly and on Day 13 (last treatment day).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was recorded with precision of 1 g at the start (Day 0) and then daily for 4 days then weekly and on Day 13 (last treatment day).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No


Sacrifice and pathology:
All surviving animals were euthanized under pentobarbital anaesthesia by exsanguination on Day 14.

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: The liver of all animals was examined including organ weight.

GROSS PATHOLOGY: Yes. The external appearance of all animals were examined including the cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs were observed macroscopically.

HISTOPATHOLOGY: No
Statistics:
Statistical analysis was performed using the statistical program package of SAS 9.3. The normality and heterogenicity of variance between groups was checked by sharpio-Wilk and Levene tests using the most appropriate data format. Where both tests show no significant heterogeneity, an Anova/Ancova (one-way analysis of variance) test was carried out. f the obtained result waspositive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control. For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used. If the group sizes were larger, than the Chi-squared test was used, identifying differences of <0.05, <0.01 or <0.001 as appropriate.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed during the study
Mortality:
no mortality observed
Description (incidence):
No mortality was recorded in the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test item-related adverse effects were observed on body weight and body weight gain in both males and females receiving 13000 ppm test item in the diet.

Low and mid dose group male lost weight on the first day however followed by a weight gain similar to control animals for the remaining days (those animals receiving 4500 ppm made a complete recovery on Day 2, whereas those receiving 13000 ppm in the diet remained ~7% below control body weight).

The females receiving 13000 ppm in the diet initially lost weight but did not make a sustained recovery until Day 4; their weight gain from Day 7 was similar to that of the controls, remaining ~10% (p<0.01) below control body weight.

Statistically significant decreases in body weight were recorded from Day 2 to Day 9 (p<0.05) for males receiving 13000 ppm in the diet and, for females, during the whole treatment period (p<0.01). A statistically significantly decreased body weight gain was observed on Day 1 in males receiving either 4500 of 13000 ppm in the diet (p<0.01) and Day 2 (males receiving 4500 ppm in the diet; p<0.01). Overall decreased body weight gain was observed on the males receiving 13000 ppm in the diet (p<0.01, by -31.5%). Statistically significantly decreased body weight gain was observed on Day 1 and by the end of the treatment period in females receiving 13000 ppm in the diet (p<0.01).

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test item-related significant effect was observed in males and females receiving 13000 ppm in the diet; since most of the effect was seen in the initial part of the study, it is most probably due to a reduced palatability of the test item-containing diets.
Statistically significantly decreased food consumption was observed through the whole treatment period for the male receiving 13000 ppm in the diet (p<0.01). Statistically significantly decreased food consumption was observed sporadically in the females receiving 13000 ppm in the diet, and with overall effect on the whole treatment period (p<0.01).
Food efficiency:
no effects observed
Description (incidence and severity):
There was no test item related effect on food conversion efficiency in any of the dosed groups.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A test item-related effect was observed on the liver weight, which was also related to body weight, in females receiving 13000 ppm in the diet.
A statistically significant (p<0.05) increase in liver weight of ~12% was observed in females receiving 13000 ppm in the diet.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item related macroscopic necropsy observations were present in all terminal animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
other: MTD
Effect level:
ca. 13 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

  

Table 2 . Analytical Results

Date of analysis

Nominal

concentration

(ppm)

Measured

concentration with 95%

confidence interval

(ppm)

Percentage of the

nominal

concentration

(%)

RSD%

(%)

30 October 2021

(Before the 1st

treatment)

Control

not detected

-

-

1300

1187 ± 24

91

1.6

4500

4203 ± 42

93

0.8

13000

12567 ± 340

97

2.2

QC – Low

100

0.3

QC – High

100

0.2

  

Table 3. Summary of Selected body weight parameters

Parameters

Dose groups

 

Control

1300 ppm

4500 ppm

13000 ppm

 

Male, Body weight on Day 14 (g)

424.5

414.8

418.7

395.7

NS

difference (%)

-2.3

-1.4

-6.8

 

Male, Body weight gain Day 0-7 (g)

50.7

44.7

46.8

23.7**

DU

difference (%)

-11.8

-7.6

-53.3

 

Male, Body weight gain Day 7-14 (g)

40.8

38.2

37.7

39.7

NS

difference (%)

-6.5

-7.8

-2.9

 

Male, Body weight gain Day 0-14 (g)

91.5

82.8

84.5

63.3**

DN

difference (%)

-9.5

-7.7

-30.8

 

Female, Body weight on Day 14 (g)

248.8

238.8

238.7

223.7**

DN

difference (%)

-4.0

-4.1

-10.1

 

Female, Body weight gain Day 0-7 (g)

22.5

16.2

12.2

0.3**

DN

difference (%)

-28.1

-45.9

-98.5

 

Female, Body weight gain Day 7-14 (g)

12.7

9.3

12.8

9.7

NS

difference (%)

-26.3

1.3

-23.7

 

Female, Body weight gain Day 0-14 (g)

35.2

25.5

25.0

10.0**

DN

difference (%)

-27.5

-28.9

-71.6

 

 

Notes: Data (group mean values, n=6) were rounded to one decimal place.

NS: Statistically not significant when compared to control, DU: Dunn 2 sided test,

Statistical significance compared to control: ** = p<0.01, DN: Dunnett 2 sided test, Bold letter: data out of the historical control range

 

  

Table 4. Daily food consumption values

 

Parameters

Dose groups

Control

1300 ppm

4500 ppm

13000 ppm

 

Male, Food consumption (Day 0-7)
(g/animal/day)

33.10

33.00

32.81

24.76

NS

difference (%)

-0.3

-0.9

-25.2

 

Male, Food consumption (Day 7-14)
(g/animal/day)

34.29

34.00

33.67

30.50**

DN

difference (%)

-0.8

-1.8

-11.0

 

Male, Food consumption (Day 0-14)
(g/animal/day)

33.69

33.50

33.24

27.63**

DN

difference (%)

-0.6

-1.3

-18.0

 

Female, Food consumption (Day 0-7)
(g/animal/day)

22.76

21.36

21.05

15.40**

DN

difference (%)

-6.2

-7.5

-32.3

 

Female, Food consumption (Day 7-14)
(g/animal/day)

21.12

20.74

19.98

17.55**

DN

difference (%)

-1.8

-5.4

-16.9

 

Female, Food consumption (Day 0-14)
(g/animal/day)

21.94

21.05

20.51

16.48**

DN

difference (%)

-4.1

-6.5

-24.9

 

 

Notes: Data (group mean values, n=6) were rounded to one decimal place.

Statistical significance compared to control: ** = p<0.01, DN: Dunnett 2 sided test 

 

Table 5. Summary of organ weight parameters

Parameter

Dose groups

Control

1300 ppm

4500 ppm

13000 ppm

Males

Liver/body weight (%)

4.487

4.299

4.521

4.705

(difference %)

-4.2

0.8

4.9

Females

Liver/body weight (%)

3.810

3.917

4.174

4.287*

(difference %)

2.8

9.6

12.5

 

Notes: Data (group mean values, n=6) were rounded to two decimal places. HC: Historical control.

Statistical significance compared to control: * = p<0.05, DN: Dunnett 2 sided test

 

Conclusions:
In conclusion, based on this 14-day Dose Range Finding (DRF) toxicity study where 1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DECYL AND OCTYL TRIESTERS was administered by pelleted diet to Wistar rats for 14 consecutive days: the dose level of 13000 ppm (977 mg/kg bw/day) was considered not to cause severe toxicity. The effects on food intake, body weight and on liver weight seen in rats receiving 13000 ppm in the diet may have been due to poor palatability, for the most part. There were no significant effects at 4500 ppm (410 mg/kg bw/day) or 1300 ppm (120 mg/kg bw/day). Therefore, a dose of 1000 mg/kg bw/day (13000 ppm) will be used as highest dose for the OECD 421.
Executive summary:

The objective of the study was to obtain preliminary information on the toxicity of the test item when administered daily to groups of 4 male and 4 female Wistar rats. The test item was administered at a constant concentration in pelleted diet at three dose levels for up to 14 days.

The study was conducted to 1) determine the correct dose levels, 2) investigate possible palatability effect of the test item, and 3) determine if dietary dosing can be used for a subsequent OECD No. 421 study [5]. It was considered that dietary dosing might be a better application approach than gavage for further studies, especially in the following OECD 443. Control animals were treated with vehicle only (non-treated diet). Male and female Wistar rats (8 weeks old animals) were treated in the study for 14 consecutive days as shown below. The first day of dosing was designated as Day 1 for each animal. The animals were treated for 14 days with test item in concentrations of 0, 1300, 4500 and 13000ppm in dose groups of control, low dose, mid dose and high dose, respectively.

The general health status and clinical observations were performed twice daily. Body weight and food consumption were measured for all animals and for body weight measurement, prior to scheduled necropsy on Day 14 (not fasted). Gross macroscopic examination was performed at necropsy in each animal. Selected organs were weighed.

Results:

The concentration of the test substance in the diets were found to be within the acceptable ranges and considered suitable for the study purposes. There was no mortality in the study and no clinical signs were observed. An initial body weight loss was observed in the animals receiving 13000 ppm in the diet. Rats receiving the High dose (13,000 ppm) had a near normal body weight gain from Day 2 (males), or from Day 7 (females); both males and females receiving the High dose (13,000 ppm) were ~7% and ~10% below control body weight after 14 days. In animals receiving 13000 ppm in the diet, test item-related decreased food consumption was observed in both sexes, mainly in the first 7 days. There were no effects evident at necropsy. In comparison to Controls, terminal body weights of animals were statistically lower in the females receiving the High dose.

1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DECYL AND OCTYL TRIESTERS was administered by diet to Wistar rats for 14 consecutive days at dose levels of 1300 (males: 118.3 mg/kg bw/day, females 121.6 mg/kg bw/day), 4500 (males: 404.8 mg/kg bw/day, females 415.7 mg/kg bw/day) and 13000 ppm (males: 981.2 mg/kg bw/day, females 971.0 mg/kg bw/day). There was no mortality in the study and no clinical signs were observed. An initial body weight loss was observed in the 13000 ppm groups, High dose males had a near normal weight gain from Day 2, females from Day 7; the High dose groups were ~7% and ~10% below control body weight after 14 days. At 13000 ppm, test item related decreased food consumption was observed in both sexes, mainly in the first 7 days. There were no effects evident at necropsy. Statistically significant increase in the body weight related liver weight was observed in High dose females.

In conclusion, based on this 14-day Dose Range Finding (DRF) toxicity study in which 1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DECYL AND OCTYL TRIESTERS was administered by pelleted diet to Wistar rats for 14 consecutive days: the dose level of 13000 ppm (977 mg/kg bw/day) was considered not to cause severe toxicity. The effects on food intake, body weight and on liver weight seen in rats receiving 13000 ppm in the diet may have been due to poor palatability, for the most part. There were no significant effects in rats receiving either 4500 ppm (410 mg/kg bw/day) or 1300 ppm (120 mg/kg bw/day). Therefore, a dose of 1000 mg/kg bw/d (13000 ppm) should be used as highest dose for the OECD 421 study.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2013 to March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 41-43 days
- Weight at study initiation: 191.7 - 222.6g (males), 152.4 - 178.5g (females)
- Fasting period before study: no
- Housing: 5 of one sex in clear polysulphone solid bottomed cages measuring 59.5x38x20 cm (Code 1354 G, Techniplast Gazzada S.a.r.l., Varese, Italy)
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedoly S.r.l., Settimo Milanese (MI), Italy) ad libitum
- Water: drinking water ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2013-05-30 (allocation of animals) To: 2013-10-03 (last day of necropsy)
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was suspended in the vehicle. The formulations were prepared daily. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification given
- Concentration in vehicle: 10, 40 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in RTC Study no. 77280 in the range from 10 to 200 mg/mL. Linearity, accuracy and precision were within the limits for suspensions (r > 0.98; accuracy 95-105 %; precision CV< 5 %).
Stability after 24 hours at room temperature was verified in the range from 10 to 200 mg/mL in RTC study no. 77280. Suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (90 %-110% for concentration and CV<10% for homogeneity). Results obtained at the end of the stability tests were still within the acceptability limits.
The proposed formulation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits for concentration (85-115 %) and homogeneity (CV<10 %).
Samples of the formulations prepared on Weeks 1 and 13 were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV<10% for homogeneity).
Duration of treatment / exposure:
13 consecutive weeks followed by a recovery period of 4 weeks for 5 males and 5 females from the control and high dose group
Frequency of treatment:
once a day, 7 days a week
Remarks:
Doses / Concentrations:
50 mg/kg/day (low dose)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/day (medium dose)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day (high dose)
Basis:
actual ingested
No. of animals per sex per dose:
10, 5 additional animals per sex for the satellite groups (control and high dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected in consultation with the study sponsor based on information from preliminary studies
- Rationale for selecting satellite groups: no rational given
- Post-exposure recovery period in satellite groups: 4 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (morality, clinical signs)
- Time schedule:
Mortality check: early each working day and again in the afternoon, at weekends and Public Holidays the second check were done at approx. mid-day
Clinical signs: daily, once before commencement of treatment and once during the study (performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions)

DETAILED CLINICAL OBSERVATIONS AND NEUROTOXICITY ASSESSMENT: Yes
- Time schedule: weekly, once before commencement of treatment and once a week thereafter
- Neurotoxicity assessment: animals were examined in an open arena for a minimum of 3 minutes, observed parameters: removal from cage, handling reactivity, lachrymation, palpebral closure, salivation, piloerection, rearing, spasms, myoclonia, mobility impairment, arousal (animal activity), vocalisation, stereotypies, unusual respiratory pattern, bizarre behaviour, urination, defecation, Tremors, gait (one of the following options: normal, ataxia, hunched, pronation forlimbs drag, hindlimbs drag)

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy

FOOD CONSUMPTION:
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes, by means of an ophthalmoscope, and by a slitlamp microscope after the instillation of 0.5% Tropicamide
- Time schedule for examinations: prior to commencement of treatment, re-examination during week 13 of treatment
- Dose groups that were examined: prior treatment: all groups, re-examination: control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13 of treatment and during week 4 of the recovery period
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 10 each sex from all groups during week 13, from all surviving animals of the recovery groups (control, high dose group)
- Parameters examined: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eocinophils, basophils, monocytes, large unstained cells), platelets, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13 of treatment and during week 4 of the recovery period
- Animals fasted: Yes
- How many animals: 10 each sex from all groups during week 13, from all surviving animals of the recovery groups (control, high dose group)
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, trigycerides, phosphorous, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: overnight during week 13 of treatment and during week 4 of the recovery period
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes and water bottles were removed, each animal received approx. 10 mL/kg bw of drinking water by gavage
- Parameters examined: appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood, sediment (obtained from centrifugation at approx. 3000 rpm for 10 min, examined microscopically for: epithelial cells leucocytes, crystals, spermatozoa and precursors, other abnormal components)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during weeks 12/13 of treatment and once during week 4 of recovery
- Dose groups that were examined: all dose groups, all animals from recovery groups (control and high dose group)
- Battery of functions tested: sensory activity (auditory, visual and propriocetive stimuli), grip strength, motor activity (measured over a period of 5 min/animal)

OTHER: Oestrous cycle and spermatogenic cycle
From the first day of Week 12 and up to the end of the treatment period, vaginal smears were taken daily in the morning. The vaginal smear data were examined to determine potential anomalies of the oestrous cycle.
A detailed qualitative evaluation of testes was performed on all control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS-H stained sections were used to identify the spermatogenic stages.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table below)
Samples of all tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethanol)

HISTOPATHOLOGY: Yes (see table below)
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides of main group animals were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed on all animals in the control and high dose groups killed after 13 weeks of treatment.
The examination was performed as detailed below:
- Tissues specified in table from all animals in the control and high dose groups killed at the end of the 13 weeks of treatment.
- Tissues specified in table from all animals killed or dying during the treatment period.
- All abnormalities in all main phase groups.
The examination was then extended to include the liver from all animals of low and mid dose groups killed after 13 weeks of treatment and those of the recovery groups (control and high dose).
Statistics:
For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated. Statistical analysis of histopathology findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
considered unrelated to treatment
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
considered of no toxicological importance
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Changes no longer observed at the end of the recovery period, therefore not considered to be adverse.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
mild treatment related findings in the liver of both sexes, and full reversible, therefore not considered to be adverse.
Details on results:
CLINICAL SIGNS AND MORTALITY
One female receiving 200 mg/kg/day was sacrificed for humane reasons (damaged hind limb) on Day 82 and one male of the same group was found dead on Day 40. The cause of death of this animal is not known.
Clinical signs in surviving animals were limited to rales and/or dyspnoea in a single female animal from Day 42 up to the end of the study.

BODY WEIGHT AND WEIGHT GAIN
No significant differences in body weights and body weight changes were noted between treated animals and controls during treatment and recovery periods.

FOOD CONSUMPTION
No treatment-related changes were observed in either sex during the experimental period.

OPHTHALMOSCOPIC EXAMINATION
No significant findings were detected at the ophthalmic examinations performed during the study.

HAEMATOLOGY
Dosing phase: Neutrophilia was recorded in some animals dosed with 500 mg/kg/day, males being more sensitive than females. Neutrophils mean group values were increased by 87% in males and 16% in females. Large unstained cells were also increased in males receiving 200 mg/kg/day and 500 mg/kg/day, with no dose-relation (32% and 23 %, respectively). The reason for this change is unclear. The statistically significant differences between control and treated animals recorded for mean corpuscular haemoglobin concentration in both sexes and eosinophils in females were of minimal severity and/or not dose- or sex-related, therefore considered unrelated to treatment.
Recovery phase: Neutrophilia was completely recovered in animals of both sexes after 4 weeks of recovery.

CLINICAL CHEMISTRY
Dosing phase: In general, changes of liver/metabolic parameters were recorded in animals dosed with 500 mg/kg/day and in males receiving 200 mg/kg/day. In particular, males dosed with 500 mg/kg/day showed increases of alkaline phosphatase, alanine aminotransferases, cholesterol, triglycerides and creatinine and decreases of bilirubin and glucose. Those receiving 200 mg/kg/day showed most of the above findings, often with less severity. Females from the same groups showed some of the above changes, such as increases of alkaline phosphatase, alanine aminotransferases, creatinine and decreases of glucose and bilirubin (see table #1 below). The other statistically significant changes recorded, such as: increase of protein, albumin and sodium in males, decrease of chloride and increase of sodium in females, were of minimal severity and/or not dose-related, therefore considered of no toxicological importance.
Recovery phase: The changes recorded during the dosing phase showed an almost complete reversibility. Total bilirubin and glucose were still slightly lower than control in some treated animals. However, due to the low severity and the absence of other related changes, the above findings were considered of no toxicological importance (see table #2 below).

URINALYSIS
No changes were recorded.

NEUROBEHAVIOUR
No differences between treated animals and controls, which could be considered of toxicological relevance, were observed at evaluations of sensory reaction performed at the end of treatment.
A slight reduction of grip strength was observed in the high dose animals of both sexes at the end of treatment and recovery periods. However, due the low magnitude, to the high individual variability of the measured data and to the absence of other correlated signs this variation was not considered of toxicological relevance.
Motor activity measurements performed at the end of the treatment or recovery periods did not show any toxicologically significant differences between treated animals and controls.

ORGAN WEIGHTS
A slight increase in the absolute weight of the liver was observed at the end of the treatment period in the males dosed at 200 and 500 mg/kg/day (+9% and +8 %, respectively) and in the females dosed at 500 mg/kg/day (+19 %, significant at statistical analysis). The relative weights of the liver were also significantly increased, at statistical analysis, in the mid- and high dose males (+6% and +9 %) and in the females from all treated groups (+9 %, +9% and +22 %). No other significant changes were observed at the end of treatment.

GROSS PATHOLOGY
No apparent tretament-related changes were noted. All observed changes were considered as incidental findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Final sacrifice: Treatment-related findings were found in the liver of both sexes treated with the high dose, and in the males treated with the mid-dose. The changes included: diffused centrilobular hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia and relative reduction in the cytoplasmic pallor (i.e., glycogen). The hypertrophy was of mild degree in males and of minimal degree in females, treated with the high dose, and of minimal degree in the males treated with the mid-dose.
All other observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions.
Recovery sacrifice: No treatment-related changes were noted

OTHER FINDINGS
Spermatogenic cycle: Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. No treatment-related changes were seen in the testes at spermatogenic staging.
Oestrus cycle: No significant differences in oestrous cycle were observed between treated and control animals.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
not specified
Table #1: Clinical chemistry in dosing phase (control data expressed in absolute values and
data from treated groups as percentage differences with controls)
Treatment AP ALT BIL CHOL TRI GLU CREA
[mg/kg/day] [U/l] [U/I] [mg/dl] (mg/dl] [mg/dl] [mg/dl] [mg/dl]
0 (control) males 229.74 37.29 0.053 69.07 44.67 136.86 0.337
50 11 -5 -25 6 45 -10* 9**
200 65** 6 -25 17** 24 -13** 10**
500 241** 94** -51** 15** 76** -12** 17**
0 (control) females 175.99 33.39 0.072 96.48 35.19 112.37 0.403
50 9 9 -11 -4 -6 -4 7
200 38** 8 -6 -7 1 -8* 1
500 100** 46** -33 -1 6 -9* 8*
Table #2: Clinical chemistry in recovery phase (control data expressed in absolute values and
data from treated groups as percentage differences with controls)
Treatment AP ALT BIL CHOL TRI GLU CREA
[mg/kg/day] [U/l] [U/I] [mg/dl] [mg/dl] [mg/dl] [mg/dl] [mg/dl]
0 (control) males 172.02 46.80 0.08 69.30 61.82 158.12 0.34
500 -8 -13** -30 1 6 -7 -6
0 (control) females 120.10 44.20 0.090 87.36 73.80 120.74 0.404
500 -8 -10 4 -1 25 -8 3
* Indicates group mean is significantly different from control at level p < 0.05
** Indicates group mean is significantly different from control at level p < 0.01
Conclusions:
Signs of treatment-related effects of the test item were observed in males and females dosed at 200 and 500 mg/kg/day. These effects included some changes in clinical pathology parameters and some post mortem findings. A main target organ, the liver, was identified on the basis of the organ weights data and histopathological findings. However, these changes were mild and fully reversible. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum. As such, the hepatic effects were not considered to be adverse.
No significant changes were observed in the animals dosed at 50 mg/kg/day. It can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 500 mg/kg/day. In the study there were not seen any indications for reproductive effects, investigated were spermatogenic staging, oestrus cycle, weights of ovaries and testes, including microscopic and macroscopic observations.
Executive summary:

The toxicity of 1,2,4-Benzenetricarboxylic acid, decyl octyl ester was investigated in Sprague Dawley rats after daily oral administration at dose levels of 50, 200 and 500 mg/kg/day for 13 weeks and recovery from any treatment-related effects during a recovery period of 4 weeks.

One female receiving 200 mg/kg/day was sacrificed for humane reasons on Day 82 for a damaged hind limb and one male of the same group was found dead on Day 40. The cause of this last death cannot be clearly established.

No significant signs of toxic or neurotoxic effects were seen during the “in-life” phase of the study.

No lesions were recorded at ophthalmological examination.

No significant differences in oestrous cycle were observed between treated and control animals, no treatment-related changes were seen in the testes at spermatogenic staging.

Neutrophilia was recorded in both male and female rats dosed with 500 mg/kg/day.

Changes of liver/metabolic parameters (increases of alkaline phosphatase, alanine aminotransferase, cholesterol, triglycerides and creatinine and decreases of bilirubin and glucose) were recorded in animals dosed with 500mg/kg/day and in males receiving 200 mg/kg/day. The majority of the above changes were reversible at the end of the recovery period.

No effects in body weight or body weight changes were seen in any of the treated groups of rats.

The absolute and relative liver weights were slightly but significantly increased in the mid- and high dose males and in all treated females. This change was correlated with the hepatocytic hypertrophy seen at the end of treatment in males from these dose groups and in the high dose females. No changes were observed at the end of the 4 week recovery period.

Minimal to mild centrilobular hepatocytic hypertrophy was observed at microscopic examination in the males dosed at 200 and 500 mg/kg/day and in the females dosed at 500 mg/kg/day. These changes were no longer present at the end of recovery. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum (see Maronpot et al, 20106). No other changes were noted in the hepatocytes (i.e., degeneration and/or necrosis) and, therefore, the changes were considered as potentially adaptive.

No treatment-related changes were seen in the testes at spermatogenic staging.

On the basis of the above mentioned results, the liver was the main target organ. However, the changes observed at 200 and 500 mg/kg/day were minimal to mild and fully reversible. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum. As such, the hepatic effects were not considered to be adverse. No significant changes were observed in the animals dosed at 50 mg/kg/day. In the study there were not seen any indications for reproductive effects, investigated were spermatogenic staging, oestrus cycle, weights of ovaries and testes, including microscopic and macroscopic observations.

It can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 500 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A key study is identified. This study was conducted according to OECD TG 408 in accordance with GLP. It is a sub-chronic study using Sprague-Dawley rats where the test substance was administered daily in corn oil at concentrations of 50, 200 and 500 mg/kg/day for 13 weeks. The NOAEL was determined to be 500 mg/kg bw/day based on treatment related effects at 200 and 500 mg/kg/day. The main target organ was identified as the liver based on organ weight data and histopathological findings. These changes were noted as mild and fully reversible. In addition the morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of the endoplasmic reticulum, such effects were not considered to be adverse.

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

In a subchronic OECD 408 study, groups of male and female Sprague Dawley rats were dosed with 0, 50, 200 or 500 mg/kg/day 1,2,4-Benzenetricarboxylic acid, decyl octyl ester in in corn oil using a dose volume of 5mL/kg for a period of 90 days. Additional satellite group of recovery animals were included to understand the post-exposure recovery period in control and Group 4 (500 mg/kg/day) animals.


Signs of treatment-related effects of the test item were observed in males and females dosed at 200 and 500 mg/kg/day. These effects included some changes in clinical pathology parameters and some post mortem findings. The liver was identified as the main target organ on the basis of the organ weight data and histopathological findings. However, these changes were mild and fully reversible. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum. As such, the hepatic effects were not considered to be adverse. No significant changes were observed in the animals dosed at 50 mg/kg/day. It can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 500 mg/kg/day. In the study there were not seen any indications for reproductive effects, those investigated were spermatogenic staging, oestrus cycle, weights of ovaries and testes, including microscopic and macroscopic observations.In a subacute oral toxicity study in rats some treatment-related effects were evident in males and to a minor extent in females at the high dose level of 1000 mg/kg/d. The adrenals were identified as the main target organs, based on the post-mortem examination. The observed effects were completely reversible over a 2-week recovery period in the high dose animals. Only mild effects were observed in the animals (essentially males) dosed at 300 mg/kg/d, therefore this dose is expected to be NOAEL. No effects were observed at the low dose level (NOEL = 100 mg/kg/d). In the subacute 28 -day study, a dose of 500 mg/kg bw/d was not used but adverse effects were seen at 1000 mg/kg bw/d (LOAEL). Therefore for repeated dose toxicity, it can be concluded in summary that the NOAEL is 500 mg/kg bw/d.

Justification for classification or non-classification

The results of the repeated dose oral toxicity studies show that 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters does not warrant classification according to Regulation (EC) No 1272/2008.