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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-20 to 2018-05-08
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:
Diethyl succinate
EC Number:
204-612-0
EC Name:
Diethyl succinate
Cas Number:
123-25-1
Molecular formula:
C8H14O4
IUPAC Name:
1,4-diethyl butanedioate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt lot No.: T101217268
- Manufacturing date: 25 Sep 2017
- Expiration date of the lot/batch: 25 Sep 2018

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: 8 - 9 weeks
- Weight at study initiation: Males: 275 - 310 g; Females: 207 - 240 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: 19 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:
mortality observed, non-treatment-related
Description (incidence):
One male treated at 1000 mg/kg bw/day found dead on Day 19. No clinical signs or other signs of toxicity were observed prior to death and necropsy followed by microscopic pathology of tissues/organs did not reveal any relevant findings permitting a cause of death to be defined. The cause of death
was thus considered incidental.
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: Statistically significant differences in monocyte and basophil numbers were noted between control and males treated at 100 and/or 300mg/kg bw/day. Prothrombin time was increased in females treated at 100 mg/kg bw/day. These findings were not dose-related and were therefore
considered to be incidental.
Recovery phase: No changes were recorded.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
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 decrease observed in landing footsplay values of males treated at 300 mg/kg bw/day was not dose-related and therefore considered to be without toxicological relevance. A slight, but statistically significant, reduction in motor activity was oberved in males treated at 1000 mg/kg bw/day and considered to be incidental due to a single animal showing a very low value.
Recovery phase: No significant changes were observed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Treatment phase: No treatment-related changes were observed in absolute and relative (% to body weight) organ weights in all treatment groups of both sexes, when compared to controls. Those variations between control and treated animals that were seen were considered to be within the physiological range of Sprague Dawley rats.
Recovery phase: No relevant changes were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted. Observed changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley rats of the same age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All findings were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley rats of the same age.
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 Effect Level (NOEL) was determined to be 1000 mg/kg bw/day for male and female rats
Executive summary:

The oral toxicity of diethyl succinate 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 Effect Level (NOEL) is 1000 mg/kg bw/day.