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Repeated dose toxicity: oral

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short-term repeated dose toxicity: oral
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
Limit test:

Test material


Test animals

Details on test animals and environmental conditions:
- Source: Charles River Italia S.p.A.
- Age at study initiation: 10-11 weeks (7-8 weeks at delivery)
- Weight at study initiation: 221 - 234 g for males and 160 - 182 g for females (upon delivery)
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5x38x20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polycarbonate cages measuring 42.5x26.6x18 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
After mating the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages measuring 42.5x26.6x18 cm for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.
- Diet: ad libitum
- Water :ad libitum
- Acclimation period: 13 days

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

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on oral exposure:
The required amount of 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched was suspended in the vehicle (corn oil) and brought to the final volume appropriate for each concentration (concentrations of 10, 31.25 and 100 mg/mL).
The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.

- Justification for use and choice of vehicle (if other than water): corn oil was a suitable vehicle
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity). Results of the analyses were within the limits of acceptance.
The stability was found to be 24 hours at room temperature in the concentration range of 10 to 100 mg/mL.
Samples of the formulations prepared on Week 1 and last week of treatment were also analysed to check the concentration. Results of the analyses were within the limits of acceptance.

Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method, in the range from 5 to 200 mg/mL. The software used for this activity was Analyst 1.5.2 (Ab Sciex).
Duration of treatment / exposure:
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.

Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum.
Frequency of treatment:
once daily
Doses / concentrations
Doses / Concentrations:
0, 40, 125, 400 mg/kg bw/day
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels had been selected in consultation with the Sponsor and were based on the results of a 14-day oral range-finding study with groups of four male and female rats, in which all animals had to be sacrificed in moribund condition on day 3 of the study at a daily dose of 1000 mg/kg bw/day. The dose of 300 mg/kg bw/day was tolerated. Based on these results, the dose of 400 mg/kg bw/day was selected as the high dose for the present study.

- Rationale for animal assignment (if not random): random
Positive control:


Observations and examinations performed and frequency:
Throughout the study, all animals were checked early in each working day and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
All clinical signs were recorded for individual animals.
Once before commencement of treatment and at least once daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination.
Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period following allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group, under condition of food deprivation.

The blood samples collected were divided into tubes as follows:

EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests
No anticoagulant for hormone assay

Red blood cell count
Red blood cell distribution width
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

Coagulation test
Prothrombin time
Activated partial thromboplastin time

Blood samples for animal nos. 93810066 and 93810068 were not analysed since the volumes were insufficient.
The blood sample for animal no. 93810005 was not analysed since it was coagulated.

Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Inorganic phosphorus
Total bilirubin
Total cholesterol
Bile acids
Total protein
A/G Ratio

Values of gamma-glutamyltransferase below the limit of quantification are reported in the appendix as NT (Not Taken).

Hormone determinations

Prior to necropsy, blood samples (approximately 1.2 mL) were taken from the abdominal vena cava of the same animals and under the same conditions mentioned in section 4.4 and from 5 male and 5 female spare untreated rats from the same batch.
Blood samples were collected into tubes without anticoagulant, centrifuged and the serum obtained was divided in several aliquots and frozen at -80°C for possible hormone determinations (T3, T4). These measurements were not performed, since no compound-related effects on the thyroids were observed.

Functional Observation Battery Tests

Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination.
Each animal was removed from the home cage and observed in an open arena.
The tests included 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, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.
Animals were examined in an open arena for a minimum of three minutes. Observed parameters, described by an evaluation scale, are indicated below:

Removal (from cage): Easy, Difficult, Very difficult
Handling reactivity: Normal, Slow, 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 posture (Slight, Moderate, Severe)
Forelimbs drag (Slight, Moderate, Marked)
Hindlimbs drag (Slight, Moderate, Marked)

All observed parameters are reported in a group incidence table. Individual data are not included in this report. Data are reported until Week 6 of the study (all females). All other data are not tabulated in this report but archived together with all raw data.

Grip strength and sensory reactivity to stimuli

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order.
For males the tests were performed the day before necropsy and for females on Day 3 post partum.

Motor activity assessment (MA)

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for 60 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order.
For males the tests were performed the day before necropsy and for females on Day 3 post partum.
Sacrifice and pathology:

Parental animals were killed by exsanguination under isofluorane anaesthesia.
Pups were euthanised (Day 4 post partum) by intraperitoneal injection of Sodium Thiopenthal.

Parental Males

The males were killed after the mating of all females (after 32-33 days of treatment).

Parental Females

The females with live pups were killed on Day 4 post partum.
One female with total litter loss was killed on the day of the occurrence of total litter loss (Day 2 post partum).
The females which did not give birth 25 days after positive identification of mating were killed shortly after.


The clinical history of the males and females of the parental generation 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 (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.

All females were examined also for the following:

a) external and internal abnormalities;
b) number of visible implantation sites (pregnant animals);
c) number of corpora lutea (pregnant animals).

Uteri of apparently non-pregnant females or uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.

All pups found dead in the cage were examined for external and internal abnormalities.
All live pups sacrificed at termination were examined for external abnormalities and sex confirmation by gonadal inspection.

Tissues fixed and preserved

Samples of all the tissues (all parental animals) listed in the table (ü) were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, Harderian glands, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination

The tissues required for histopathological examination are listed in the table (ü). After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides of all males in the control and high dose groups were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

In the first instance the examination was restricted as detailed below:

a) Reproductive organs: cervix, clitoral gland, ovaries, uterus and vagina from all parental females
b) Reproductive organs: coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes from all parental males
c) Tissues specified in the table (ü) from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term
d) Tissues specified in the table (ü) from all animals dying during the treatment period
e) All abnormalities in all groups

The examination was extended to include the lungs, including mainsten bronchi (both sexes), thymus and jejunum (females only) from the remaining animals of the control and high dose groups. The examination was also extended to the thymus (females only) from all other dose groups.
Other examinations:
Organ weights

From all animals completing the scheduled test period, the organs indicated in the table (ü) were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test when ‘n’ was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Results and discussion

Results of examinations

Details on results:
Two cases of premature death occurred during the study. One male of the control group was found dead on Day 2 of treatment but the cause of death could not be determined. One female of the high dose group was found dead on Day 3 of treatment and the cause of death was considered as a consequence of mis-dosing.

Two females were found not pregnant: one control and one mid-dose.
One high dose female was sacrificed for total litter loss on Day 2 post partum.
The number of females with live pups on Day 4 post partum was: 9 in each of the control and mid-dose groups (125 mg/kg bw/day), 10 in the low dose group (40 mg/kg bw/day) and 8 in the high dose group (400 mg/kg bw/day).

Salivation and decreased muscle tone were the main clinical sign observed in all high dose males throughout the study and in all high dose females during post coitum and post partum periods. Salivation was considered most probably related to the taste of the compound and therefore considered not to be adverse. Decreased muscle tone occurred as an isolated finding, since no alterations were observed in the functional observation battery tests, especially in the grip strength and foot splay tests, and was therefore not considered to be toxicologically relevant. In addition, rales were also occasionally noted in 5 out of 10 treated males and females of the high dose group. This clinical sign was not considered to be compound-related due to its transient appearance. Due to the low incidence, other clinical signs recorded were considered of no toxicological importance

Males of the high dose group showed a slight decrease in body weight during the first week of administration (approximately 13 g). Body weights were slightly, but statistically significant reduced (approximately 8%) in the high dose males at the end of Weeks 3 and 4 of treatment, when compared to controls. However, these changes were mainly attributable to one male (no. 93810066), which had clinical signs, particularly lower body weights, and histopthological changes suggestive for mis-dosing.
No difference in body weight was detected in treated females during the whole study, when compared to controls.
Body weight gain of treated animals did not show differences of toxicological importance, when compared to controls.
Terminal body weight was slightly decreased in treated males with statistical significance in the high dose group (10%). No differences were observed in females.

Food consumption was unaffected by treatment in both sexes during the study.

No changes of toxicological significance were observed.
The statistically significant differences between control and treated animals (haemoglobin, haematocrit, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red blood cells distribution width, eosinophils in males, platelets, lymphocytes and basophils in females) were of low magnitude and/or not dose-related, and therefore considered unrelated to treatment.

A lower activated partial thromboplastin time was recorded in males dosed with 125 and 400 mg/kg/day (19% and 21%, respectively) and in females treated with 400 mg/kg/day (20%).
Due to the low severity, changes were considered to be of no toxicological significance.

Higher values of alkaline phosphatase (71%), alanine aminotransferase (61%) and phosphorus (35%) were observed in females dosed with 400 mg/kg/day.
Glucose was higher in treated males, showing no dose-relationship (approximately 37%).
Due to the absence of other clinical pathology findings and/or related histopathological changes, the above-mentioned differences were considered of no toxicological relevance.

Neurotoxicity assessment (removal of animals from the home cage and open arena)
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
Motor activity and sensory reaction to stimuli measurements recorded at the end of treatment were comparable between control and treated groups in animals of both sexes.

Some statistically significant differences were noted in the absolute and/or relative organ weight of both sexes.

In males:
- Lower absolute seminal vesicles weight in high dose males (22%).
- Higher relative adrenals (27%), brain (15%), kidneys (15%) and testes (13%) weights in high dose males.

In females:
- Lower absolute and relative ovaries weights in low, mid- and high dose females (20%, 19% and 26% respectively for the absolute weight and 21%, 21% and 25% respectively for the relative weight).
- Lower absolute and relative thymus weight in high dose females (28% and 27% respectively).

The increased relative organ weights and the lower absolute seminal vesicle weights in males were considered to be due to the reduced terminal body weight. None of the above differences was accompanied by histological findings. Therefore, they were considered of no toxicological importance.

Detailed macroscopic observations were reported for individual animals in the study.
Microscopic evaluation was performed on 5 randomly selected animals from each of Groups 1 and 4 (males and females). In addition, the reproductive organs from all male and female animals from all groups, as well as all macroscopic abnormalities from all groups were examined histologically.
Potential treatment-related lesions were detected in the lungs, including bronchi, of both sexes and in the jejunum and thymus of the females only. The lungs were suggested to be checked in the rest of the animals of both sexes in Groups 1 and 4. Moreover, the thymus and jejunum were suggested to be checked in the rest of the females in Groups 1 and 4, while in all female animals from Groups 2 and 3, only thymus was examined.
For ease and clarity of reporting, only tissues with diagnoses have been reported in the incidence tables.

Unscheduled death

Two cases of premature death occurred during the study.
The list of the findings observed in the individual animals is as follows:
Animal no. 93810016 (male control group):
Macroscopically, the following changes were noted: caecum: distended; lungs: incomplete collapse and dark color; thymus: red color; prostate and seminal vesicle: small size.
Microscopically, the changes were as follows: liver: inflammatory cell foci; thymus and lungs: congestion. The cause of death could not be determined.

Animal no. 93810069 (female high dose group):
Macroscopically, the following changes were noted: muzzle: red staining; stomach and jejunum: red mucosa; liver: a single pale raised area; thymus: red color; trachea: a single abnormal area; lungs: incomplete collapse and dark read areas; and pancreas: pallor.
Microscopically, the changes were as follows: stomach: multifocal erosions in the glandular mucosa; liver: congestion; thymus: mild atrophy; jejunum: autholysis; trachea: mild mucosal necrosis associated with mild submucosal subchronic inflammation; lungs: bronchi - mild necrosis, associated with mild submucosal subchronic inflammation and presence of inflammatory exudate in lumen. The changes in the trachea and lungs are suggested to be related to the mis-dosing of the test item. Other changes noted in the lungs consisted of oedema and congestion and are considered as agonal changes.
The cause of death is considered as related to mis-dosing of the test item.

Terminal kill

Macroscopic observations

No treatment-related changes were noted.
Even though there was an increased incidence of reduced size of thymus when comparing the incidence in the high dose females (n=6) to the control group (n=1), histopathological evaluation did not confirm a treatment effect.

Microscopic observations

No treatment-related findings were noted.

In a single male rat (no. 93810066) from the high dose, the bronchi and the alveoli in the centriacinar areas contained mucus-like fluid. This change was associated with goblet-cell metaplasia of the epithelium lining the bronchi, peribronchial fibrosis, presence of polymorphonuclear cells and histiocytes within the mucus.
The lung changes are suggested to be related to mis-dosing of the test item.
All other observed changes are considered as incidental findings, unrelated to treatment with the test compound.
The incidence of thymic atrophy in the female groups was as follows: mild grade: 9/10; 10/10; 9/10 and 8/10, respectively in the control, low, mid and high dose. A single case of moderate degree of thymic atrophy was noted in the female high dose group. Due to almost comparable incidence of thymic atrophy seen in the control and treated groups, this lesion is not considered as related to treatment.

Spermatogenic 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.
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.

Effect levels

Dose descriptor:
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no systemic toxicity was observed up to the highest dose level tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The toxic effects on rats of both sexes after repeated dosing with2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated.

The dosages used were 0 (control), 40, 125 and 400 mg/kg/day.

One control male and one high dose females died during the study, the deaths being not compound-related.

One control female and one mid-dose femalewere found not pregnant at necropsy.

In addition, one high dose female was sacrificed for total litter loss on Day 2 post partum.

The number of females with live pups on Day 4 post partum was: 9 in each of the control and mid-dose groups, 10 in the low dose group and 8 in the high dose group.

Decreased muscle tone was observed in males and females of the high dose group.This finding was considered not to be toxicologically relevant, since no effects were seen in the functional observation battery tests, especially in the grip strength and foot splay tests.Males of the high dose showed a slight body weight loss during the first week of the study, and body weights remained slightly decreased compared to the other groups throughout the study. However, these changes were mainly attributable to one male, which was probably mis-dosed. No findings of toxicological significance were noted during the in-vivo phase, both in male and female animals.

No toxicologically relevant differences were found in terms of mating performance and reproductive parameters.

No significant differences were found on litter data parameters and sex ratio.

No toxicologically significant changes were observed in clinical pathological investigations.

No treatment-related changes were observed both in male and female animals at post mortem macroscopica nd microscopic examinations and organ weight analysis.

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both systemic toxicity and reproduction/developmental toxicity was considered to be 400 mg/kg/day for males and females.



Applicant's summary and conclusion