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Toxicological information

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:
31 January - 03 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1987 according to the notification of November 21, 2003 by Ministry of Health, Labor and Welfare (No. 1121002), Ministry of Economy, Trade and Industry (No. 2) and Ministry of Environment (No. 031121002)
Deviations:
no
Principles of method if other than guideline:
None.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Light brown slightly viscous liquid
Details on test material:
- Name of test material (as cited in study report): N-[2-piperazin-1-yl)ethyl]C18-unsatured-alkylamide
- Substance type: Light brown slightly viscous liquid
- Physical state: viscous liquid
- Purity: 94%
- Lot/batch No.: S-000923
- Expiration date of the lot/batch: 16 March 2017
- Storage condition of test material: At room temperature in the dark under nitrogen
- Purity/composition correction factor required: No
- Hygroscopic: No
- Volatile: No
- Reactivity: Reactive to oxygen
- Test substance handling: Flush container with nitrogen after handling
- Specific gravity / density: 0.96 g/mL
- Stability at higher temperatures: Yes, maximum temperature: 100°C (at least several hours)
- Stability in Propylene glycol: Stability for at least 5 hours at room temperature and 8 days in the refrigerator is confirmed over the concentration range 3 to 30 mg/mL.

Test animals

Species:
rat
Strain:
other: Wistar (Han)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 168 grams; females: 135 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17.8-22.9°C
- Humidity: 24-86%
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31 January - 03 May 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
From Day 1 to 21, formulations (w/w) were prepared daily within 5 hours prior to dosing. From Day 22 onwards, formulations (w/w) were prepared for a maximum of 8 days prior to dosing. Formulations were homogenized to visually acceptable levels. Adjustment was made for density of the test substance and specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe and on information provided the sponsor.

DOSE VOLUME:
5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The delegated phase was performed by the Principal Investigator for Formulation Analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions and stability over 8 days in the refrigerator in the dark were also determined (highest and lowest concentration).The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
91 days (males) or 92 days (females). Animals were dosed up to the day prior to necropsy.
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 50, 150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 28-day study with N-[2-piperazin-1-yl)ethyl]C18-unsatured-alkylamide (information provided by the sponsor).
Positive control:
Not required.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. Clinical observations (clinical signs and arena) were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals (The results of a previous 28-day study conducted with this test substance did not provide information on a possible peak effect of occurrence of signs after dosing; information supplied by the sponsor). The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT:
Weekly.

FOOD CONSUMPTION:
Weekly.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: At pretest: all animals (including spare animals), at Week 13 : Groups 1 and 4. Since no treatment-related ophthalmologic findings were noted in Week 13, the eyes of the rats of Groups 2 and 3 were not examined.

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS:
No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: at Week 12 : Groups 1 and 4
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.

OTHER:
Estrous cycle determination: All females had a daily lavage from Day 71 up to and including Day 91 to determine the stage of estrous.
Sacrifice and pathology:
GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. All animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
- Dose groups that were examined: Groups 1 and 4 (According to test guidelines)
- lungs, duodenum, jejunum and mesenteric lymph nodes of all males and females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- additional slids of the testes from all group 1 and 4 animals for spermatogenesis staging.


Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
based on subjective appraisal
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY AND CLINICAL SIGNS
No mortality occurred during the study period. No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among all animals at 50 and 150 mg/kg during the larger part of the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance. Incidental findings that were noted included scabs, wounds, diarrhoea, rales, alopecia and a missing upper incisor. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No toxicologically significant ophthalmology findings were noted. Incidental ophthalmology findings at pretest (all animals examined) and/or in Week 13 (Group 1 and 4 animals examined) consisted of focal corneal opacity, pinpoint corneal opacities, focal corneal oedema, haemorrhage from the hyaloid vessel and persistent pupillary membrane. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes in haematological parameters were considered to be of no toxicological significance as they occurred in the absence of a dose-related trend and remained (essentially) within the range considered normal for rats of this age and strain. These changes were noted in males only and consisted of a lower haemoglobin level at 15, 50 and 150 mg/kg, higher relative eosinophilic counts, lower mean corpuscular haemoglobin concentration (MCHC) and lower activated partial thromboplastin time (APTT) at 50 mg/kg, and higher prothrombin time (PT) at 150 mg/kg.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a dose-related trend and remained (essentially) within the range considered normal for rats of this age and strain. These changes consisted of a higher alanine aminotransferase activity (ALAT) in males at females at 150 mg/kg, higher alkaline phosphatase activity (ALP) in males at 50 mg/kg, lower total protein, urea and chloride level in males at 150 mg/kg, higher sodium level in males at 15 and 50 mg/kg, lower total bilirubin level in females at 15 and 50 mg/kg, and higher sodium and lower potassium level in females at 15 mg/kg.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals. Motor activity was similar between the 150 mg/kg group and control group, and both groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
No toxicologically significant changes were noted in organ weights and organ to body weight ratios. The statistically significant lower prostate and seminal vesicle weight (also for prostate to body weight ratio) at 150 mg/kg had no microscopic correlate and remained within the range considered normal for rats of this age and strain. These changes were therefore considered not to be toxicologically relevant.

GROSS PATHOLOGY
No toxicologically relevant necropsy findings were noted. Reddish foci on the glandular mucosa of the stomach in 3/10 males and red discolouration of the mandibular lymph nodes of 4/10 females at 150 mg/kg had no treatment-related microscopic correlate. The incidence of other necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY
The following microscopic findings were considered to be related to treatment:
- Lung: increased incidence of alveolar macrophages in males at 50 mg/kg (minimal degree) and in males and females at 150 mg/kg (up to slight degree),
- Small intestines: microvesicular vacuolation of enterocytes up to slight degree in 9/10 males and in 4/10 females at 150 mg/kg. In most cases the duodenum and/or jejunum were affected. The size of the vacuoles varied from one-quarter (halfway the villi) up to one-half of the nuclear size (on the top of the villi). Vacuoles were located supra-nuclear, at the luminal side.
- Mesenteric lymph node: increased incidence and severity of cellularity of the paracortical areas. Incidence and severities consisted of 1/10 males (minimal) and 1/10 females (minimal) treated at 0 mg/kg, 3/10 males (minimal) and 2/10 females (minimal) treated at 15 mg/kg, 4/10 males (3 minimal, 1 slight) and 3/10 females (minimal) treated at 50 mg/kg and 5/10 males (1 minimal, 4 slight) and 6/10 females (4 minimal, 2 slight) treated at 150 mg/kg.

Spermatogenic staging profiles were normal in all Group 1 and 4 males.

All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

OTHER FINDINGS
Estrous cycle determination: All females showed a normal (regular) estrous cycle of 4 days during the period in which estrous cycle length was determined (Day 71 up to and including Day 91).

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

ANALYSIS OF DOSE PREPARATIONS

No test substance was detected in the Group 1 formulations. The concentrations analyzed in the formulations of Group 2, 3 and 4 in Weeks 1, 6 and 13 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). One of the accuracy samples collected from the Group 2 formulation used for dosing in Week 6 did not show a signal. The reasons for this could not be determined. Based on overall analytical results, an adequate assessment of the analytical results could still be made. The formulations of Group 2 and 4 in Weeks 1, 6 and 13 were homogeneous (i.e. coefficient of variation ≤10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours and stored under nitrogen in the refrigerator in the dark for at least 8 days (i.e. relative difference ≤ 10%).

Applicant's summary and conclusion

Conclusions:
In this 90-day oral repeated dose toxicity study with rats, the NOAEL was determined to be 50 mg/kg bw/day.

Executive summary:

Subchronic oral toxicity of N-[2-piperazin-1-yl)ethyl] C18-unsatured-alkylamide was evaluated in a 90-day study in rat following daily dosing by gavage according to OECD 408.

The dose levels for this 90-day oral gavage study were selected at 0, 15, 50 and 150 mg/kg, based on results of a 28-day study.

The test substance, formulated in propylene glycol, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Chemical analyses of formulations were conducted during the study to assess accuracy, homogeneity and stability over 5 hours at room temperature and 8 days in the refrigerator.

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 12; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

 

Results:

Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 5 hours at room temperature and 8 days in the refrigerator.

Treatment-related findings were confined to histopathological lesions.

Increased incidence of alveolar macrophages in the lungs was observed in males at 50 mg/kg and both sexes at 150 mg/kg (up to slight degree). When considering all lung observations, showing especially high incidences of inflammation in the controls, the resulting overall picture does not really indicate a biologically significant difference. Taking into account the low severities of alveolar macrophages after 90 days of treatment, and given that the increase in incidence was slight, this change was considered not adverse in nature.

In addition, microvesicular vacuolation of enterocytes of small intestines in both sexes at 150 mg/kg (up to slight degree) and a dose-related increased incidence and severity of cellularity of the paracortical areas of the mesenteric lymph node in both sexes was noted. This latter finding is most probably related to the changes observed in intestinal enterocytes, since other primary and secondary lymphoid organs did not show treatment related changes. The paracortical area is also known to have an immunologic function in T-cell response. For the findings in the intestines and draining lymph nodes the toxicological relevance remains unknown, and as such these changes were considered to be adverse in nature.

No toxicologically significant changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, clinical laboratory investigations, macroscopic examination and organ weights).

There were no indications of possible reproductive toxicity based on the parameters determined in this study: All females showed a normal (regular) estrous cycle of 4 days during the period in which estrous cycle length was determined (Day 71 up to and including Day 91), and histopathological examination of the male and female reproductive organs did not show treatment-related lesions.

 

Conclusion:

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for N-[2-piperazin-1-yl)ethyl]C18-unsatured-alkylamide of 50 mg/kg was established.