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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28. Feb. 2005 - 05. Jul. 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Labelled : Isononylbenzoat
Batch Number : 04/3945-3947
Retest date : 12 July 2005
Received from : Oxeno GmbH
Date received : 21 February 2005
Amount received : Approximately 2 kg
Description : Clear liquid
Storage at RTC : Room temperature
RTC reference number : 9428

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 27-29 days
- Weight at study initiation: males: 93.4 - 109.0 g; females: 91.0 - 109.8 g
- Fasting period before study:
- Housing: 5 of one sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Code 1354 G, Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Each cage tray held absorbent paper which was inspected and changed at least 3 times a week.
- Diet : 4RF21, Mucedola S.r.l., Settimo Milanese (MI), Italy ad libitum
- Water : ad libitum
- Acclimation period: 17 days


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


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of Benzoic Acid Isononylester was suspended in the vehicle. The formulation was prepared daily (concentrations of 3.75, 37.5 and 75 mg/ml). Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil is a standard vehicle in this kind of study
- Concentration in vehicle: 3.75, 37.5 and 75 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg bw
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of the study, analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stabilityof the formulation was satisfactory. Samples of the formulations prepared in weeks 1 and 13 of the study were also analysed to
check the homogeneity and concentration
Duration of treatment / exposure:
All animals were dosed once a day, 7 days a week, for a minimum of 13 consecutive weeks followed by a recovery period of 4 weeks for 5 males and 5 females from groups 1 and 4. Animals in the main phase were dosed up until the day before necropsy. No treatment was given to animals during the recovery period.
Frequency of treatment:
All animals were dosed once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 150, 300 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
In addition, groups 1 and 4 included an extra 5 animals per sex to be killed after 4 weeks of recovery.
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a 4 week repeat dose study.
- Rationale for animal assignment: random
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale: random

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Examination of individual animals for signs of reaction to treatment was carried out daily prior to dosing, immediately after dosing and then at approximately 1 and 2 hours after dosing for the first 9 days of treatment. The number and timing of these daily observations
was reviewed by the Study Director at the end of the first week of treatment and the number of observations was reduced, the observation immediately following dosing was no longer performed from day 10 of treatment onwards.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once per week from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena for a period of 3 minutes. Observed parameters, described by an evaluation scale, are
indicated below:
Removal (from cage): Easy, Difficult, Very difficult
Handling reactivity: Normal, Slight, Moderate, Marked
Lachrymation: Absent, Slight, Marked
Palpebral closure: Absent, Slight, Moderate, Marked
Salivation: Absent, Slight, Marked
Piloerection: Absent, Present
Rearing: Absent, Intervals of number of times (i.e. 1–3, 4-7, 8-10)
Spasms: Absent, Tonic spasms, Clonic spasms, Tonic-clonic spasms
Myoclonia: Absent, Present
Mobility impairment: Absent, Slight, Moderate, Marked
Arousal (animal activity): Very slow, Slow, Normal, Moderate, Marked
Vocalisation: Absent, Present
Stereotypies: Absent, Present
Unusual respiratory pattern: Absent, Present
Bizarre behaviour: Absent, Present
Urination: Absent, Intervals of number of times (i.e. 1–3, 4-6)
Defecation: Absent, Intervals of number of times (i.e. 1–3, 4-6)
Tremors : Absent, Present
Gait (one of the following options): Normal
Ataxia (Slight, Moderate, Marked)
Hunched (Slight, Moderate, Severely)
Pronation
Fore limbs drag (Slight, Moderate, Marked)
Hind limbs drag (Slight, Moderate, Marked)
BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD EFFICIENCY:
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.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined: Both eyes of all animals assigned to the study were examined just prior to the commencement of treatment by means of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide (Visumidriatic®, Visufarma, Rome, Italy). In the first instance the eyes of all animals from high dose and control groups were re-examined during week 13 of treatment. Since no changes correlated to the treatment were observed, no further examination was necessary.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 13 from the retro-orbital sinus, prior to necropsy from the abdominal vena cava of the same rats
from each group, during week 4 of recovery
- Anaesthetic used for blood collection: Yes, light ether anaesthesia week 13, prior to necropsy under isofluorane anaesthesia
- Animals fasted: Yes
- How many animals: first 10 surviving male and female animals of each group
- Parameters checked
Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count (not performed as no signs of anaemia or polychromasia were observed)
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
Abnormalities of the blood film
Platelets
Prothrombin time

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
- How many animals: first 10 surviving male and female animals of each group
- Parameters checked:
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: during week 13
- 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
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:
Epithelial cells
Polymorphonuclear leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during week 12 of treatment and once during week 4 of recovery an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength was also performed. The motor activity (MA) of all animals was measured once during week 12 of treatment and once during week 4 of recovery by an automated activity recording. Measurements were performed using a computer generated random order.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity /


OTHER:
Sacrifice and pathology:
All animals were killed by exsanguination under isofluorane anaesthesia and subjected to necropsy, supervised by a pathologist. The clinical history of each animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved. The tissues required for histopathological examination are Abnormalities, Adrenal glands, Aorta, Bone marrow (from sternum), Brain, Caecum, Colon, Duodenum, Epididymides, Eyes, Esophagus, Femur with joint, Heart, Ileum (including Peyer’s patches), Jejunum, Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes - cervical, Lymph nodes - mesenteric, Mammary area, Ovaries, Oviducts, Pancreas, Parathyroid glands, Pituitary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal column, Spinal cord, Spleen, Stomach, Testes, Thymus (where present), Thyroid, Trachea, Urinary bladder, Uterus - cervix
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 using a pooled error variance. The homogeneity of the data was verified by Bartlett’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 from the actual values in the computer without rounding off. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

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 specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths occurred during the study. No signs of reaction to treatment were revealed during daily pre- and post-dose observations nor weekly functional observation tests.

BODY WEIGHT AND WEIGHT GAIN
There were no significant differences in body weights between treatment groups during the
study.

OPHTHALMOSCOPIC EXAMINATION
No lesions of toxicological significance were detected at the ophthalmic examination performed during week 13 of treatment.

HAEMATOLOGY
Following 13 weeks of treatment, haematological analyses revealed a reduction in white blood cells (with the exception of neutrophils) in mid- and high dose males. A slight anaemia and thrombocytopenia were also observed in some males from the high dose group. However, since values were within the range of our historical data, all the above mentioned changes were considered of no toxicological importance. At the end of the treatment-free period, the white blood cell count of the high dose groups was similar to controls. The slight anaemia was still present in males but it was considered
incidental and values were within the range of our historical dada.

CLINICAL CHEMISTRY
A slight increase in triglycerides was observed in treated males, in a dose-related trend, and in some females receiving 300 mg/kg/day. Some values were outside the range of our historical data. However, since values outside the range of historical data were present also in the control animals and there were no other related changes in the metabolic profile and/or at histopathological examination, change in triglycerides cannot be definitively considered of toxicological significance. The other biochemical changes observed in a number of animals from the high dose group (decreases in gamma-glutamyltrasferase, albumin and calcium increases in sodium and potassium) were also considered incidental, within our historical data, therefor without toxicological significance. At the end of the recovery phase, no alterations were observed which could be attributed to treatment with the test item.

URINALYSIS
No significant alterations in urine parameters were observed at the end of the treatment and recovery period.

NEUROBEHAVIOUR
Neurotoxicity tests and motor activity measurements taken at the end of treatment and recovery did not show changes attributable to the test item.

ORGAN WEIGHTS
Statistically significant increases in relative and/or absolute liver and kidney weights were noted in high dose males and females, as well as mid-dose females. In addition, a statistically significant increase in absolute thymus weight was recorded in high dose males. At the end of the treatment-free period, the changes in liver, kidney and thymus weights were no longer significant, indicating a full recovery at the high dose level.
All further changes (increase in absolute testes weight in low dose males and decrease in relative brain weight in mid- and high dose females) at the end of treatment and recovery (adrenal and heart weights in females and testes weights in males) were not considered of toxicological importance as no dose relationship could be identified during the treatment period or the findings were inconsistent between sexes or considered spontaneous in origin at the end of recovery.

GROSS PATHOLOGY
No macroscopic findings were described, that could be considered correlated with the administration of the test item.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic evaluation has been performed on control and high dose group animals, on the liver of all animals and on all abnormalities in all groups.
For ease and clarity of reporting, only tissues with findings are presented in the incidence tables. No change was observed in the examined tissues/organs, which could be considered treatment-related.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The changes reported, including the neoplasm described in a female receiving 15 mg/kg/day, were considered to be either expression of spontaneous pathology, commonly seen in this species under our experimental conditions or incidental in origin.

HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant changes were observed

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mortality: No deaths occurred during the study.

Pre- and post-dose observations and Clinical signs: No signs of reaction to treatment were revealed during daily pre- and post-dose observations nor weekly functional observation tests. On day 57 of treatment, red staining was noted on a high dose male cage tray. This finding was incidental in nature and not considered treatment-related.

Motor activity and sensory reaction to stimuli: Neurotoxicity tests and motor activity measurements taken at the end of treatment and

recovery did not show changes attributable to the test item.

Body weight: There were no significant differences in body weights between treatment groups during the study.

Food consumption: Food consumption was comparable between groups throughout the treatment and recovery phase.

Ophthalmoscopy: No lesions of toxicological significance were detected at the ophthalmic examination performed during week 13 of treatment.

Haematology: Following 13 weeks of treatment, haematological analyses revealed a reduction in white blood cells (with the exception of neutrophils) in mid- and high dose males. A slight anaemia and thrombocytopenia were also observed in some males from the high

dose group. However, since values were within the range of our historical data, all the above mentioned changes were considered of no toxicological importance.

At the end of the treatment-free period, the white blood cell count of the high dose groups was similar to controls. The slight anaemia was still present in males but it was considered incidental and values were within the range of our historical dada.

Clinical chemistry: A slight increase in triglycerides was observed in treated males, in a dose-related trend, and in some females receiving 300 mg/kg/day. Some values were outside the range of our historical data. However, since values outside the range of historical data were present also in the control animals and there were no other related changes in the metabolic profile and/or at

histopathological examination, change in triglycerides cannot be definitively considered of toxicological significance.

The other biochemical changes observed in a number of animals from the high dose group (decreases in gamma-glutamyltrasferase, albumin and calcium increases in sodium and potassium) were also considered incidental, within our historical data, therefore without

toxicological significance. At the end of the recovery phase, no alterations were observed which could be attributed to

treatment with the test item.

Urinalysis: No significant alterations in urine parameters were observed at the end of the treatment and recovery period.

Terminal body weight and organ weights: At the end of the treatment period, slightly higher terminal body weights were recorded in all

treated groups in both sexes, when compared with controls, attaining statistical significance in mid-dose females. This finding was not considered treatment-related since no dose dependence could be ascribed. At the end of the recovery period, no statistically significant changes were reported between the high dose group and that of the control. Statistically significant increases in relative and/or absolute liver and kidney weights were noted in high dose males and females, as well as mid-dose females. In addition, a statistically significant increase in absolute thymus weight was recorded in high dose males. At the end of the treatment-free period, the changes in liver, kidney and thymus weights were no longer significant, indicating a full recovery at the high dose level.

All further changes (increase in absolute testes weight in low dose males and decrease in relative brain weight in mid- and high dose females) at the end of treatment and recovery (adrenal and heart weights in females and testes weights in males) were not considered of toxicological importance as no dose relationship could be identified during the treatment period or the findings were inconsistent between sexes or considered spontaneous in origin at the end of recovery.

Macroscopic observations: Macroscopic observations have been reported for individual animals in the study. No macroscopic findings were described, that could be considered correlated with the administration of the test item.

Microscopic observations: Microscopic evaluation has been performed on control and high dose group animals, on the

liver of all animals and on all abnormalities in all groups. No change was observed in the examined tissues/organs, which could be considered treatment-related. The changes reported, including the neoplasm described in a female receiving 15 mg/kg/day,

were considered to be either expression of spontaneous pathology, commonly seen in this species under our experimental conditions or incidental in origin.

Applicant's summary and conclusion

Conclusions:
Following 13 weeks of treatment, no toxicologically relevant changes were observed in body weight, food consumption, clinical observations (reaction to treatment, clinical signs, neurotoxicity assessment, motor activity) and at clinical pathology investigations. The changes in organ weights observed at the post-mortem examinations [increased weights at the high dose level in both sexes (liver and kidney) as well as at the mid-dose level in
females (liver), in addition to high dose level males (thymus)], were reversed at the end of the treatment-free period, indicating a complete resolution after sufficient time. However, no corresponding lesion was observed at the histopathology examination.
On the basis of these results, the No Observed Adverse Effect Level (NOAEL) in this study is considered to be 300 mg/kg/day.
Executive summary:

The toxicity of Benzoic Acid Isononylester when given by oral administration (gavage) to rats for 13 consecutive weeks at dosages of 15, 150 and 300 mg/kg/day has been investigated.

Following 13 weeks of treatment, no toxicologically relevant changes were observed in body weight, food consumption, clinical observations (reaction to treatment, clinical signs, neurotoxicity assessment, motor activity) and at clinical pathology investigations. The changes in organ weights observed at the post-mortem examinations [increased weights at the high dose level in both sexes (liver and kidney) as well as at the mid-dose level in

females (liver), in addition to high dose level males (thymus)], were reversed at the end of the treatment-free period, indicating a complete resolution after sufficient time. However, no corresponding lesion was observed at the histopathology examination.

On the basis of these results, the No Observed Adverse Effect Level (NOAEL) in this study is considered to be 300 mg/kg/day.