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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:
01 Sep 2017 - 17 Dec 2018
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):N-(3-Aminopropyl)-imidazol
- Physical state: Liquid/ colorless, clear
- Analytical purity:99.9 area-% (project No. 17L00242)

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL:
Batch identification:C520/090/2017/F3
Purity: 99,9% area%
Manufacturing date: 29 Jun 2017

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- SOURCE: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at the beginning of the administration period: 42 ± 1 days
- Fasting period before study: All remaining animals were sacrificed after a fasting period period (withdrawal of food) of at least 16 hours.
- Housing: The animals were housed together (5 animals per cage) in polysulfonate cages.
- Diet (e.g. ad libitum) Kliba maintenance diet mouse/rat “GLP, Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): The drinking water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24°C
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
other: highly deionized water
Details on oral exposure:
The test substance was applied as an aqueous solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration.Then, ultrapure water (synonym: highly deionized water) was filled up to the desired volume and subsequently homogenized with a magnetic stirrer. The test-substance preparations were produced at least twice a week and stored at room temperature. The administration volume was 10 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory. The study was carried out in compliance with the Principles of Good Laboratory Practice.The stability of the test substance in ultrapure water (synonym: highly deionized water) at room temperature for a period of 7 days was proven in a comparable batch .Homogeneity was verified in 3 samples in the highest and lowest concentration (was used as a concentration control at the same time at the beginning and towards the end of the administration period; additional concentration control analyses were done in the mid concentration
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
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:
10
Control animals:
yes, concurrent vehicle
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.The following parameters were examined:Abnormal behavior when handled ,Fur , Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Impairment of gail, Lacrimation, Palpebral closure, Exophthalmus, Feces (appearance/ consistency), Urine, Pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

Food consumption: YES
- Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day. Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Prior to the start of the administration period on day -1 the eyes of all animals and on study day 87 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: (Day 94 male and Day 95 female)
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: (Day 94 male and Day 95 female)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: (Day 87)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined :all animals
- Battery of functions tested: sensory activity / grip strength / motor activity assessment
- FOB(Functional observational battery): Home cage observations, Open field observations, Sensoy imotor tests, Quantitative parameters

IMMUNOLOGY: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights:
Anesthetized animals (final body weight), adrenal glands (fixed),Brain, Epididymides, Heart, Kidneys, Liver, Ovaries (fixed), Spleen, Testes, Thymus (fixed)Thyroid glands (with parathyroid glands; fixed), Uterus with cervix

HISTOPATHOLOGY: Yes
All gross lesions , Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum , Cervix , Coagulating glands, Colon, Duodenum , Epididymides, EsophagusEyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes, mesenteric, Lymph nodes, axillary, Mammary gland (female),Ovaries, Pancreas, Parathyroid glands, Peyer’s patches, Pituitary gland, Prostate ,Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina
Other examinations:
SPERM PARAMETERS:
motile sperms, total spermatids/gram testis, total sperms/gram cauda epididymis, abnormal sperms
Statistics:
Body weight/ change:
A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.

Rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity, estrous cycle.
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.

Blood parameters:
For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided), for the hypothesis of equal medians.

Urinalysis parameters (apart from urine volume, specific gravity, color and turbidity:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. In case of exactly the same numbers of the dose group and the control, no statistical test is performed.

Urine volume and specific gravity:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians.

Weight parameters:
Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each test group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related, adverse effects were obtained in test groups 1 and 2 (100 and 300 mg/kg bw/d).
Semi-closed eyelids shortly after application were observed in all male and 7 female animals of test group 3 (1000 mg/kg bw/d) on several days shortly after treatment. The finding occurred for the first time in female animal No. 72 on study day 27. For male animals, the finding was firstly observed in animal Nos. 35 and 37 on study day 33. The occurrence of this finding was assessed to be treatment-related and adverse. Salivation shortly after application was observed in all male and female animals of test group 3 (1000 mg/kg bw/d) on several days of the study. From the temporary, short appearance immediately after dosing it was concluded that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. Nasal discharge was observed in male animal No. 38 on study day 25 and female animal No. 73 on study day 41. Respiration sounds were detected in female animal No. 73 on study day 41 and in female animal No. 80 on study day 86. The findings were considered to be related to treatment but assessed to be non-adverse as they did not occur in regular intervals but only spontaneously. Mass palpable through skin was observed in female animal No. 62 of test group 2 (300 mg/kg bw/d) from study day 91 onwards. Skin lesion in neck region was detected in male animal No. 3 of test group 0 (control) between study days 21 to 41. A relation to treatment was excluded.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female animal No. 72 of test group 3 (1000 mg/kg bw/d) was found dead on study day 43. According to the results obtained during histopathological examinations, a gavage error occurred. Thus, a relation to the test substance was considered to be unlikely. No further animals died prematurely in the present study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No significantly changes of mean body weight values were found in male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d). However, mean body weight change values were significantly decreased in male animals of test group 3 (1000 mg/kg bw/d) on study days 28, 49 to 70 and 91 with a maximum by -12.0% on study day 70. These changes were assessed as being related to treatment and adverse. Significant changes of mean body weight change values were neither found in male animals of test group 1-2 (100 and 300 mg/kg bw/d) nor in female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to food consumption were observed.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to water consumption were observed.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were observed. All apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.
The left pupil of female animal No. 55 of test group 1 (100 mg/kg bw/d) was not completely open after the administration with the mydriatic agent. Since the finding was recorded before treatment started, a relation to treatment was excluded.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in rats of both sexes of test group 3 (1000 mg/kg bw/d) absolute reticulocyte counts were significantly increased. Additionally, in males of the mentioned test group hemoglobin and hematocrit values were significantly lower compared to controls whereas platelet counts were significantly increased. These alterations were regarded as treatment-related and adverse.
Already in males of test group 2 (300 mg/kg bw/d) absolute reticulocyte counts were significantly higher compared to controls. However, this was the only changed red blood cell parameter among these individuals and the mean was within the historical control range (males, absolute reticulocytes 108.5-170.1 giga/L). Therefore, this change was regarded as treatment-related, but non-adverse. In females of test group 1 (100 mg/kg bw/d) mean corpuscular volume (MCV) was significantly higher compared to controls, but the value was not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.

Regarding the differential blood cell counts, in rats of both sexes of test group 3 (1000 mg/kg bw/d) absolute neutrophil cell counts were significantly increased. This was also true for the relative neutrophil counts in males of the mentioned test group. These alterations were regarded as treatment-related and adverse.
Absolute eosinophil counts were increased in males of test groups 2 and 3 (300 and 1000 mg/kg bw/d; in test group 3 not statistically significant), but they were decreased in females of test group 1 (100 mg/kg bw/d, not dose-dependently) and test group 3 (1000 mg/kg bw/d). Relative eosinophil counts were also significantly decreased in females of test group 3. Although the values in test group 3 were beyond historical control ranges (absolute eosinophils, males 0.08-0.17 giga/L; females 0.06-0.16 giga/L; relative eosinophils, females 1.3-5.7%), the sex-related contrary regulation made it highly unlikely that these changes were treatment-related. Additionally, in females of test group 3 (1000 mg/kg bw/d), relative basophil cell counts were significantly decreased, but the mean was within the historical control range (females, relative basophils 0.1-0.8%). Absolute monocyte counts were significantly lower in females of test group 1 (100 mg/kg bw/d), but this alteration was not dose-dependent. Therefore, all mentioned changes in this paragraph were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of administration period in rats of both sexes of test group 3 (1000 mg/kg bw/d), total protein, albumin, globulin and sodium values were significantly decreased whereas inorganic phosphate levels were significantly increased. Additionally, in
males of the mentioned test group, urea and potassium levels were significantly higher compared to controls and in females of test group 3 triglyceride values were significantly increased. These alterations were regarded as treatment-related and adverse.
Total protein and globulin values were already significantly decreased in males of test group 2 (300 mg/kg bw/d) and potassium levels were significantly higher in males of this test group compared to controls. However, the values of all three parameters were within historical control ranges (males, total protein 59.69-65.90 g/L; globulins 22.72-30.71 g/L; potassium 4.39-5.04 mmol/L). The same was true for significantly decreased total bilirubin values in females of test group 3 (1000 mg/kg bw/d; females, total bilirubin 1.41-2.74 µmol/L). Significantly higher triglyceride levels in males of test group 2 (300 mg/kg bw/d) as well as significantly increased chloride levels in females of test group 1 (100 mg/kg bw/d) were not dose-dependently changed. Therefore, all mentioned alterations in this paragraph were regarded as incidental and not treatment-related
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period in rats of both sexes of test group 3 (1000 mg/kg bw/d) higher incidences of triple-phosphate crystals were observed in the urine sediment. Without any histopathological correlate in the renal tract and without any other changed clinical pathology kidney parameter, this finding was regarded as treatment-related, but non-adverse. In males of test group 2 (300 mg/kg bw/d) urine specific gravity was significantly decreased, whereas in males of test group 3 (1000 mg/kg bw/d) specific gravity was significantly increased. Because of this contrary regulation, this finding was regarded as incidental and not treatment-related.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
FOB-Functional observational battery:-Grip-strength of hindlimbs was significantly decreased in male animals of test group 3 (1000 mg/kg bw/d) by -13.1%. Taking the results obtained for clinical pathology parameters as well as the impaired body weight development into account, this deviation to the control could have been interpreted as a sign of weakness. Thus, the change was considered to be related to treatment and assessed to be adverse.
Home cage observations, Open field observations, Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters: No test substance-related effects were observed for male animals of test groups 1-2 (100 and 300 mg/kg bw/d) and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/
Motor activity measurement: Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for male and female animals of test groups 1-2 (100 and 300 mg/kg bw/d). At single intervals No. 1 and No. 2, decreased values were measured for male and female animals of test group 3 (1000 mg/kg bw/d). In addition, the overall motor activity for male animals of test group 3 (1000 mg/kg bw/d) was significantly decreased. The overall motor activity for female animals of test group 3 (1000 mg/kg bw/d) was also lower compared to the control group, but not significantly. These changes were considered to be related to treatment and adverse. 
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The decrease in terminal body weight of males of test group 3 (1000 mg/kg bw/d) was regarded to be treatment-related. The increased absolute and relative liver weight in females of test group 3 (1000 mg/kg bw/d) was regarded to be treatment-related taking the changed clinical pathology parameters into account.
The increase in kidney weight of males and females of test group 3 (1000 mg/kg bw/d) did not reveal a histopathologic correlate. Also, in clinical pathology, no adverse findings were observed. The weight increase was therefore regarded to be treatment-related but not adverse. All the other weight changes did either not show a dose response-relationship (heart, thyroid gland) and/or revealed no histopathologic correlate that could explain the weight changes (all). Parameters checked in table [4] were examined.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
These macroscopic findings summarized in this table were regarded to be treatment-related. Duodenum, Forestomach, GI.stomach, mesenteric LN.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. Parameters checked in table [5] were examined Decedents: Female animal No. 72 of test group 3 (1000 mg/kg bw/d), which was found dead on study day 43, did not show any gross lesion.

Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
All findings in test group 3 (1000 mg/kg bw/d) males and females in forestomach and glandular stomach were signs of local irritation. The forestomach was more severely affected than the glandular stomach. These findings were regarded to be treatment-related.
In test group 2 (300 mg/kg bw/d) two males and one female revealed erosion/ulcer in the glandular stomach. These findings were regarded to be incidental for the following reasons: in the three cases, the finding was only a small erosion, no other findings were observed in the stomach, especially no findings were seen in the forestomach which was the main target region in test group 3 (1000 mg/kg bw/d) animals. Therefore, it was regarded as an incidental finding, which also occurs in the glandular stomach of control rats (Greaves, 2012). In the duodenum of male and female animals of test group 3 (1000 mg/kg bw/d) an increase in villous height and depth of crypts was observed and diagnosed as mucosal hypertrophy. It was regarded to be a reaction related to the findings observed in the fore- and glandular stomach and assessed to be treatment-related. Erythrocytes in the sinusoids of the mesenteric lymph nodes are most often transported via the lymph from hemorrhages in the draining area. In this case, it corresponded in test group 3 animals (1000 mg/kg bw/d), with one exception, to animals having erosion/ulcer in fore- or glandular stomach or animals showing hemorrhage in the glandular stomach. The macroscopically observed discolorations correlated with the microscopic finding. It was therefore a consequence to the stomach findings and related to treatment. The minimal accumulations of erythrocytes in each one male of test group 1 and 2 (100 and 300 mg/kg bw/d) and two females of test group 2 (300 mg/kg bw/d) were regarded to be incidental.The increase of hematopoiesis in the spleen is seen as increased demand of erythrocytes and inflammatory cells due to the erosion/ulcer and inflammatory processes in the stomach. It was regarded to be secondary to the stomach findings and treatment-related.Parameters checked in table [6] were examined.
Decedents
Female animal No. 72 of test group 3 (1000 mg/kg bw/d), which was found dead on study day 43, had inflammatory cell infiltrates in lung and trachea and necrosis within the trachea. These findings are observed regularly in correlation with a gavage error. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of N-(3-Aminopropyl)imidazole by gavage to male and female Wistar rats for 3 months caused signs of systemic toxicity at a dose level of 1000 mg/kg bw/d taking clinical pathology and pathology parameters into account.
Thus, the no observed adverse effect level (NOAEL) was 300 mg/kg bw/d for male and female Wistar rats.