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Description of key information

28 -day sub-acute toxicity: NOAEL 1000 mg/kg/day

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-19 to 2018-05-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
October 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Production lot
- Lot/batch No.of test material: TM870
- Expiration date of the lot/batch: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected as the most appropriate species being that preferred by the test guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia SpA, Calco (Lecco), Italy
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: Males: 267 - 301 g; Females: 197 - 227 g
- Fasting period before study: No, not applicable
- Housing: Group housed (5 of same sex/cage) in polysulfone cages
- Diet: Rodent diet (4RF21, Mucedela Srl) ad libitum
- Water: Municipal drinking water ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL.

VEHICLE
- Justification for use and choice of vehicle: The substance is not soluble in water therefore corn oil was used for preparing formulations appropriate for oral administration.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of formulations for concentration and homogeneity was performed twice during the study period.
Samples were taken from different places from each concentration for analysis of concentration and homogeneity. Similar sampling was undertaken from the vehicle control.
Measured concentrations were within the test facility acceptance criteria of 85 - 115% with CV < 10%.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 male/ 5 female per group
Additional satellite groups of 5 male / 5 female for control and high dose to examine effects of recovery.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for selecting satellite groups: Groups pre-assigned, animal assignment with groups random as for main study groups
- Post-exposure recovery period in satellite groups: 2 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least daily, at same time interval
- Cage side observations included: Mortality/morbidity, gross clinical signs/response to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
- Observations included: Detailed clinical examination in an open arena. Observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual
respiratory pattern).

BODY WEIGHT: Yes
- Time schedule for examinations: On the day treatment commenced and weekly thereafter.

FOOD CONSUMPTION: Yes
- Weight of food consumed by each cage of rats recorded at weekly intervals. Group mean daily intake per rat calculated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On termination of treatment and at end of Week 2 of recovery
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals
- 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, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On termination of treatment and at end of Week 2 of recovery
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: On termination of treatment and at end of Week 2 of recovery
- Metabolism cages used for collection of urine: Yes - overnight
- Animals fasted: Yes
- Parameters examined: Appearance, Volume, Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood, Sediment, obtained from centrifugation, was examined microscopically for: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during last week of exposure and during Week 2 of recovery
- Dose groups that were examined: All groups
- Battery of functions tested: Sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Detailed post mortem examination including examination of the external surface and orifices.
Organ weights - Adrenal glands, Brain, Coagulating glands, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate gland, Seminal vesicles, Spleen, Testes, Thymus, Thyroid, Uterus.
Tissues fixed and preserved: Abnormalities, Adrenal glands, Bone marrow (from sternum), Brain (cerebrum, cerebellum, medulla/pons), Caecum, Coagulating glands, Colon, Duodenum, Epididymides, Eyes, Femur with joint, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes – cervical, Lymph nodes – mesenteric, Mammary area (males and females), Ovaries, Oviducts, Parathyroid glands, Pituitary gland, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal column, Spinal cord, Spleen, Stomach, Testes, Thymus (where present), Thyroid gland, Trachea, Urinary bladder, Uterus – cervix, Vagina.

HISTOPATHOLOGY: Yes
Full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups killed on termination of treatment. After dehydration and embedding in paraffin wax, sections were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
Statistics:
Standard deviations were calculated as considered appropriate. 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 using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test
(Cochran and Cox) was applied.
Results were evaluated in comparison with values of control group (i.e. control value).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed in all male animals treated at 1000 mg/kg bw/day, generally starting from Day 20 of the treatment period (end of Week 3/Week 4). No other clinical signs were observed during the treatment and recovery periods.
No changes of toxicological significance were found at the weekly clinical examination which included an evaluation of neurotoxicity.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in any group (control, 100, 300 or 1000 mg/kg bw/day) during the course of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Neither body weight nor body weight gain were affected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no test item influence on the mean daily food consumption of male or female animals during the treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment phase: Mean corpuscular volume and mean corpuscular haemoglobin showed a statistically significant decrease in females dosed at 1000 mg/kg bw/day (4% below controls). Due to the minimal severity, these findings were considered to be of no toxicological significance.
Recovery phase: No relevant changes were noted.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment phase: Statistically significant changes of some biochemical parameters, relative to controls, were noted. These included increased alkaline phosphatase (53%) and decreased chloride (2%), protein (7%) and globulin (12%) in males dosed at 1000 mg/kg bw/day. Chloride was decreased (2%) in males dosed at 300 mg/kg bw/day. In females, decreased chloride (2%) and increased potassium (11%) were noted in the high dose group treated at 1000 mg/kg bw/day. Due to the minimal severity, these findingswere considered to be of no toxicological significance.
Recovery phase: Statistically significant differences between control and treated males were noted in calcium, sodium and phosphorus. As these were not observed at the dosing phase they were therefore considered to be incidental.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No differences between treated animals and controls, which could be considered of toxicological relevance, were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of the treatment and recovery periods.
Motor activity did not show any change of toxicological relevance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment phase: Body weight of treated animals was comparable to the concurrent control group. A mild but statistically significant increase in mean relative liver weight was noted in males and females treated at 1000 mg/kg bw/day, this increase being approximately 15% in both males and females. All other organ weight variations between control and treated animals were considered to be within the physiological range of Sprague Dawley SD rats.
Recovery phase: Organ weight deviations related to treatment were not seen. A minimal, although statistically significant, relative liver weight increase observed in the treated high dose males was considered incidental since all individual values were within the physiological range of variability.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted, following gross pathology examination at the end of the treatment and recovery phases. Those changes that were observed were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
A pale area was observed in the liver of one male treated at 1000 mg/kg bw/day which correlated microscopically with focal zonal necrosis, a finding considered incidental rather than treatment related as a result of its single occurrence and focal distribution.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the 4-week treatment period, no histopathological changes attributed to treatment were noted in the high dose animals treated at 1000 mg/kg bw/day. All observed findings were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Key result
Critical effects observed:
no
Conclusions:
The tested substance did not cause adverse effects in male or female rats after 28 consecutive days oral (by gavage) administration at 100, 300 or 1000 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/day for male and female rats
Executive summary:

The oral toxicity of triisononyl benzene-1,2,4 -tricarboxylate in Sprague Dawley SD rats, following daily oral administration at dose levels of 100, 300 and 1000 mg/kg bw/day for 4 consecutive weeks and recovery from any treatment-related effect during a treatment-free period of 2 weeks, has been investigated.

No mortality occurred during the study. Salivation was the only relevant clinical sign, observed in males treated at 1000 mg/kg bw/day. No signs indicating neurotoxic effects were seen. Body weight and food consumption were not affected by treatment.

Clinical pathology evaluations (including haematology, coagulation, clinical chemistry and urinalysis), performed both at the end of the treatment and recovery periods, revealed no relevant signs of toxicity. A mild, statistically significant, increase (+15%) in mean relative liver weight was seen in males and females treated at 1000 mg/kg bw/day. This increase was no longer observed at the end of the recovery period. The observed increase was not accompanied by corroborative histopathological changes (i.e. hepatocellular hypertrophy). Gross pathology and histopathology examination of preserved tissues/organs did not reveal any treatment related alterations.

No treatment-related effects were observed in animals dosed at 100 and 300mg/kg/day.

It was concluded that the No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg bw/day and the No Observed Effect Level (NOEL) is 300 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

Data are available on the registered substance from a sub-acute study of 28 days duration. No mortality occurred, there were no significant clinical signs nor any signs indicating neurotoxic effects of treatment. Body weight and food consumption were not affected by treatment. Clinical pathology evaluations revealed no relevant signs of toxicity. A small increase relative liver weight was seen in males and females treated at 1000 mg/kg bw/day, this no longer apparent at the end of the recovery period.The observed increase was not accompanied by corroborative histopathological changes (i.e. hepatocellular hypertrophy). Gross pathology and histopathology examination of preserved tissues/organs did not reveal any treatment related alterations. It was concluded that the No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg bw/day.

 

Data are also available from a longer duration study 90 day study on a structurally similar trimellitate, tri(2 -ethylhexyl)trimellitate (TOTM/TEHTM), which differs from the registered substance only in the length of the ester side chaines (branched C8 vs. branched C9). In this study there were no significant changes in clinical signs, body weight or food consumption. Minor changes in some blood chemistry parameters, together with organ weights suggest an effect in the liver. Microscopic pathology indicated changes to the liver and spleen which, although possibly adaptive, could be attributed to administration of the substance. The NO(A)EL in this study was considered to be 225 mg/kg/day in males & females, equivalent to 242 mg/kg bodyweight for the registered substance.

Justification for classification or non-classification

According to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification and labelling is not indicated for repeated dose toxicity, as clear functional disturbances or morphological changes were not apparent, the effects that were observed being minor, probably adaptive, changes.