Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-14 to 2018-05-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethyl benzene-1,2,4-tricarboxylate
EC Number:
219-547-3
EC Name:
Trimethyl benzene-1,2,4-tricarboxylate
Cas Number:
2459-10-1
Molecular formula:
C12H12O6
IUPAC Name:
1,2,4-trimethyl benzene-1,2,4-tricarboxylate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Production lot
- Lot/batch No.of test material: 221273406F
- Expiration date of the lot/batch: 2019-01-19

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions

Test animals

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: 210 - 236 g; Females: 183 - 216 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: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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, 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).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed during the treatment and recovery periods.

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: A statistically significant increase of monocytes was noted in females dosed at 100 mg/kg/day. A statistically significant increase of prothrombin time was noted in females dosed at 300 mg/kg bw/day. These were not dose-related and therefore considered to be unrelated to treatment.
Recovery phase: A slight decrease of leucocytes was noted in treated males. Since this finding was not observed at the dosing phase, it was considered to be unrelated to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment phase: Albumin was increased in treated females, with some dose-relation. Due to the minimal severity and the absence of other related
changes, this finding was considered to be of no toxicological relevance. Statistically significant increases of bilirubin were noted in females dosed at
100 mg/kg bw/day and of potassium in those dosed at 100 and 300 mg/kg bw/day. These were not dose-related and therefore considered to be incidental.
Recovery phase: Albumin was comparable with controls, even though values were similar to those recorded at the end of treatment. Statistically significant differences of chloride and phosphorus were noted in treated males compared with controls. As such differences were not observed during the dosing phase they were considered to be incidental.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment phase: Urinary volume was decreased in males and females dosed at 1000 mg/kg bw/day. Due to the absence of other related findings, this change was considered to be of no toxicological relevance.
Recovery phase: Urinary volume showed an almost complete reversibility in both sexes
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Treatment phase: No differences between treated animals and controls, which could be considered of toxicological relevance, were observed. A statistically significant reduction in landing foot splay, not in a dose-related manner, was noted at the end of treatment in all treated males whereas no differences were noted in landing foot splay measurements in females during treatment. Motor activity measurements did not show any toxicologically significant differences between treated animals and controls.
Recovery phase: No toxicologically significant differences between treated animals and controls were apparent.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment phase: No relevant changes were observed in absolute and relative organ weights of treated animals when compared to controls. An increased absolute and relative spleen weight was noted in one female treated at 1000 mg/kg bw/day which was considered incidental and therefore not toxicologically relevant.
Recovery phase: No relevant changes were noted.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant macroscopic changes were observed that could be considered treatment-related. An enlarged spleen was observed in one female treated at 1000 mg/kg bw/day which was considered spontaneous and incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted in animals killed at the end of the treatment period. Extramedullary haematopoiesis of spleen and liver was detected in one female treated at 1000 mg/kg bw/day but this is commonly observed in rodents as a normal component of the splenic red pulp and of the hepatic lobules. It occurs more frequently in young than in aged animals, in females than in males, and in mice than in rats. Hematopoietic cell numbers may increase above normal background due to a variety of conditions and may include increased numbers of erythroid precursors. The above mentioned findings in the spleen were correlated to the gross observations detected at post mortem (enlarged spleen). Hepatocytic vacuolation in the periportal area was also reported in the liver of the same animal. The above changes and, a number of sporadic lesions that were seen in control and treated animals, were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age of animal.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

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 trimethyl 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 clinical signs were observed in the animals during treatment or recovery periods. Neurotoxicity assessment did not reveal changes of toxicological relevance. No toxicologically significant changes in body weight and food consumption were observed. No changes of toxicological relevance or changes considered related to treatment were observed at the haematology analysis, coagulation parameter and at clinical chemistry investigations, as well as urinalysis. No treatment-related changes were detected at post mortem examination and subsequent microscopic examination of preserved tissues and organs.

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