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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Oct 2019 - 11 Feb 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2018
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2008
GLP compliance:
yes (incl. QA statement)
Remarks:
Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-methyl-N-oleoylglycine
EC Number:
701-177-3
Molecular formula:
C21H39NO3
IUPAC Name:
N-methyl-N-oleoylglycine

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD)
Details on species / strain selection:
Selected due to its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 58 days (males), 59 days (females)
- Weight at study initiation: 290.7 g - 351.7 g (males), 207.4 g - 271.5 g (females)
- Housing: 3 animals/cage in MAKROLON cages (type III plus or type IV) with granulated textured wood (Granulat A2, J. Brandenburg, Goldenstedt, Germany) as bedding material.
- Diet: Commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water in bottles, ad libitum
- Acclimatisation period: 13 days (males), 14 days (females)

DETAILS OF FOOD AND WATER QUALITY:
Periodic analysis of the food for contaminants is conducted by Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Institut für Tiergesundheit und Lebensmittelqualität GmbH (Kiel, Germany). Certificates of analysis of the composition and for contaminants are provided by the manufacturer. The drinking water is examined according to the "Deutsche Trinkwasserverordnung 2018" [German Regulations on drinking water 2018] by the Hamburger Wasser-werke, Hamburg, Germany, at least four times/year. In addition, once/year the drinking water is subjected to a bacteriological examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 15 Oct 2019 To: 11 Feb 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous hydroxylpropyl methyl-cellulose gel
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concentrations (w/v) and was administered by gavage at a constant volume of 10 mL/kg bw/day. The dose formulations were continuously stirred throughout the entire administration procedure to ensure homogeneity. The dose of the test item has been adapted to the animal's body weight daily up to and including test week 6, thereafter weekly.

VEHICLE
- Supplier: Fagron Services B.V., The Netherlands
- Lot/batch no. (if required): 18A17-B04
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method (HPLC-UV) was developed and validated in a GLP study (LPT study no. 37647). A test item formulation of 100 mg/L was homogenous and stable for at least 8 h. The following parameters were determined: linearity, accuracy, precision, sensitivity, specificity and stability. The accuracy of all formulation samples obtained during the study was well within the acceptable limit of ±15% (percentage of the actual concentration of active ingredient in the formulation samples compared to the respective nominal concentration).
Duration of treatment / exposure:
90 days (control and test groups)
90 days and 28 days post-exposure observation period (recovery control and high-dose groups)
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
The initial dose level of the high-dose group was reduced to 750 mg/kg bw as of test days 30 (males) an 29 (females) and further reduced to 600 mg/kg bw as of test days 37 (males) an 36 (female) due to observed morbidity/mortality.
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
The initial dose level of the high-dose group was reduced to 750 mg/kg bw as of test days 30 (males) an 29 (females) and further reduced to 600 mg/kg bw as of test days 37 (males) an 36 (female) due to observed morbidity/mortality.
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
The initial dose level of the high-dose group was reduced to 750 mg/kg bw as of test days 30 (males) an 29 (females) and further reduced to 600 mg/kg bw as of test days 37 (males) an 36 (female) due to observed morbidity/mortality.
No. of animals per sex per dose:
Main study: 10 (40 males and 40 females)
Recovery animals: 5 (10 males and 10 females), only control and high-dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a foregoing 28-day dose range finding study, in which animals were orally exposed to 100, 300 and 1000 mg/kg bw/day for 28 days (LPT study no. 37715). Only slightly reduced body weights and body weight gains were observed at 300 and 1000 mg/kg bw/day but no severe signs of toxicity were detected. Therefore, 100, 300 and 1000 mg/kg bw/day were selected as the dose levels for the main study.
- Rationale for selecting satellite groups: Investigation of the reversibility of any effects after a treatment-free recovery period.
- Post-exposure recovery period in satellite groups: 14 days
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Anaesthesia for blood sampling: yes, isoflurane

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily from 7:30 a.m. to 4:30 p.m., on Saturdays and Sundays from 8:00 a.m. to 12:00 a.m.
- Cage side observations include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behavior patterns.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and once/week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of group allocation, daily from the day of commencement of treatment up to and including test week 6 for dose adjustment, thereafter weekly throughout the experimental period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before first dosing, at the end of treatment period and at the end of the recovery period.
- Dose groups that were examined: performed on all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of test week 13 (main study); at the end of recovery period (recovery control and high-dose groups)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all main study and all recovery groups animals
- Parameters checked: differential blood count (relative and absolute), Erythrocytes (RBC), Leucocytes (WBC), Haematocrit value, Haemoglobin content, Platelets, Reticulocytes, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Prothrombin time (PT), Activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of test week 13 (main study); at the end of recovery period (recovery control and high-dose groups)
- Animals fasted: Yes
- How many animals: all main study and all recovery groups animals
- Parameters checked: Sodium, Potassium, Calcium, Chloride, Albumin, Total bilirubin, Total cholesterol, High density lipoprotein (HDL), Low density lipoprotein (LDL), Glucose, Total protein, Blood urea nitrogen (BUN), Creatinine, Bile acids, Aspartate aminotransferase (ASAT/GOT), Alanine amino-transferase (ALAT/GPT), Alkaline phosphatase (aP), Lactate dehydrogenase (LDH), Thyroid hormones (T3, T4, TSH)

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of test week 13 (main study); at the end of recovery period (recovery control and high-dose groups)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked : Colour and the turbidity, Volume, pH, Specific gravity, Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Hemoglobin (Hb), Nitrite

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of test week 13 (main study); at the end of recovery period (recovery control and high-dose groups)
- Dose groups that were examined: all main study and all recovery groups animals
- Battery of functions tested: sensory activity and grip strength and motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organs to be weighed: Adrenal gland, Brain, Epididymis, Heart, Kidney, Liver, Ovary, Spleen, Pituitary, Prostate and seminal vesicles with coagulating glands, Testicle, Thymus, Thyroid, Uterus (including cervix)
- Tissues to be preserved: Adrenal gland, Aorta abdominalis, Bone (os femoris with joint), Bone marrow (os femoris), Brain (cerebrum, cerebellum, medulla/pons), Epididymis, Eye with optic nerve, Heart (left and right ventricle, septum), Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches), Intestine, large (colon, rectum), Kidney and ureter, Liver (2 lobes), Lungs (with mainstem bronchi and bronchioles, Lymph node (1, cervical and 1, mesenteric), Mammary gland, Muscle (skeletal, leg), Nerve (sciatic), Oesophagus, Ovary with oviducts, Pancreas, Pituitary, Prostate and seminal vesicles with coagulating glands, Salivary glands (mandibular, parotid, sublingual), Skin (left flank), Spinal cord (3 sections: cervical, mid-thoracic, lumbar), Spleen, Stomach, Testicle, Thymus, Thyroid (including parathyroids), Tissue masses or tumours (including regional lymph nodes), Trachea (including larynx), Urinary bladder, Uterus (including cervix), Vagina

HISTOPATHOLOGY: Yes
- Animals: all main study animals of the control and high-dose groups and all animals of the recovery and high-dose groups.
- Organs and tissues to be examined: the same as summarised under 'GROSS PATHOLOGY'
Statistics:
Multiple t-test based on DUNNETT: Body weight / Haematology / Coagulation / Clinical chemistry / Relative and absolute organ weights (p <= 0.05 and p <= 0.01)
STUDENT's t-test: Body temperature / Hind leg splay / Grip strength / Spontaneous motility (p <= 0.05 and p <= 0.01)
Exact test of FISHER: Histopathology (p <= 0.05)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation and breathing sounds occurred during the treatment period in the mid- and high-dose groups. The detailed clinical observations performed once per week confirmed the clinical signs. For details please refer to Tables 2 and 3 under 'Attached background material'.
Mortality:
mortality observed, treatment-related
Description (incidence):
Deaths/sacrifices caused by local changes to the stomach occurred at the low-dose level (1 of 10 males sacrificed) and at the high-dose level (2 of 15 males and 2 of 15 females). No further deaths were noted after the dose reduction to 600 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males of the mid-dose and males and females of the high-dose groups revealed a slightly reduced body weight gain during the treatment period, resulting in a reduced absolute body weight of the animals compared to the control group. For details please refer to Tables 7, 8 and 9 under 'Attached background material'.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment with 1000/750/600 mg/kg bw/day led to a slightly decreased absolute food intake of male and female animals in several test weeks during the treatment period compared to the control group (by up to 16% in males and 20% in females). A significant decrease of the corresponding relative food intake was only observed in test week 1 (by 9% in males and 17% in females compared to the control group). For details please refer to Tables 10 and 11 under 'Attached background material'.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
For details please refer to Table 16 under 'Attached background material'.
Haematological findings:
no effects observed
Description (incidence and severity):
For details please refer to Table 12 under 'Attached background material'.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes were noted in males and females of the mid- and high-dose groups at the end of the treatment period: decreased plasma levels of total cholesterol and HDL, increased plasma levels of LDL and increased enzyme activities of ALAT and aP. In addition, decreased plasma levels of total protein, chloride and sodium were observed for the animals of the high-dose group. For details please refer to Table 13 under 'Attached background material'.
Urinalysis findings:
no effects observed
Description (incidence and severity):
For details please refer to Table 15 under 'Attached background material'.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
For details please refer to Tables 4, 5 and 6 under 'Attached background material'.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The females of the mid- and high-dose groups revealed increased relative and absolute liver weights at terminal sacrifice. For details please refer to Tables 18 and 19 under 'Attached background material'.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment with 300 or 1000/750/600 mg/kg bw/day led to a thickened mucosa in the stomachs (partly associated with yellow or white deposits on the mucosa) of male and female animals. For details please refer to Table 17 under 'Attached background material'.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the stomach of males and females of the low-, mid- and high-dose groups squamous cell hyperplasia with hyperkeratosis, minimal to slight erosions/ulcerations and submucosal oedema was observed. Further, changes were noted in the liver of males and females treated with 300 or 1000/750/600 mg/kg bw/day in form of an increase in cytoplasmic eosinophilic granulation (resulting in an increase in overall cellular eosinophilia).
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormone (T3, T4 and TSH): Increased serum levels of T4 (thyroxine) at the end of the treatment period compared to the control animals was observed in animals of the mid- and high-dose groups. For details please refer to Table 14 under 'Attached background material'.
Details on results:
A complete recovery was noted for the following parameters during or at the end of the recovery period: all clinical signs, the food intake, the plasma levels of total protein, chloride, sodium and LDL, the serum level of T4, and the relative and absolute liver weights of the male and female animals were within the range of the control group values. The macroscopic inspection at necropsy did not reveal any visible changes related to the previous treatment with the test item. The histopathological examination of the recovery animals revealed a complete recovery of the changes noted for the liver of male and female animals.
A trend towards recovery was noted for the decreased body weight gain of male and female animals, and for the total cholesterol and HDL plasma levels of the male animals of the high-dose group. The microscopic changes noted in the forestomach of males and females in form of squamous hyperplasia were still observed in male and female animals of the high-dose group; however, at a lower severity, indicating a trend towards recovery.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Noteworthy observations of the prematurely deceased/sacrificed animals of the high dose group

Animal

no./ Sex

Noteworthy observations

71 m

(PS,

TD 36)

Clinical signs

 

Salivation (slight to moderate), breathing sounds;

no particular premortal symptoms

Body weight gain

 

Body weight gain reduced by approx. 50% compared to control group (test days 1-36)

Macroscopy

Stomach: mucosa thickened

Histopathology

 

Slight squamous cell hyperplasia with minimal hyperkeratosis in the forestomach

Animal

no./ Sex

Noteworthy observations

80 m

(PS,

TD 35

Clinical signs

 

Salivation (slight to moderate), breathing sounds;

no particular premortal symptoms

Body weight gain

 

 

Body weight loss of 22 g (i.e. 6%) from test days 22 to 29; body weight gain reduced by approx. 50% compared to control group (test days 1-29)

Macroscopy

 

 

Stomach: mucosa thickened and indurated

Intestine (small): dilated

Adrenals: enlarged

Histopathology

 

 

 

Moderate squamous cell hyperplasia with hyperkeratosis, erosions and submucosal oedema in the forestomach, and minimal hepatocellular cytoplasmic alteration (eosinophilic granulation) in the liver

87 f

(PD,

TD 28)

Clinical signs

 

Salivation (slight to moderate), breathing sounds;

no particular premortal symptoms

Body weight gain

 

 

Body weight loss of 55 g (i.e. 20%) from test days 22 to 27; body weight gain reduced by approx. 40% compared to control group (test days 1-22)

Macroscopy

 

 

 

 

 

External observations: nose haemorrhagic, abdomen

dilated

Stomach: mucosa thickened

Intestines: dilated

Adrenals: enlarged, dark red discoloured

All inner organs: autolytic

Histopathology

Assessment not possible due to severe autolysis

Animal

no./ Sex

Noteworthy observations

94 f

(PS,

TD 24)

Clinical signs

 

Salivation (slight to moderate), breathing sounds;

no particular premortal symptoms

Body weight gain

 

 

Body weight loss of 23 g (i.e. 9%) from test days 22 to 24; body weight gain reduced by approx. 40% compared to control group (test days 1-22)

Macroscopy

 

 

 

 

 

 

 

 

 

 

 

External observations: abdomen dilated

Stomach: cardia region: mucosa with white deposits,

enlarged; dilated

Intestines: dilated

Duodenum: mucosa with white deposits and thickened

Liver (left medial lobe): periphery with white focus

(approx. 0.5 x 6 mm, sharp-edged)

Liver (left lateral lobe): white focus (approx. 2 x

12 mm)

Liver (right medial lobe): white focus (approx. 0.5 x

8 mm)

Adrenals: enlarged, dark red discoloured

Histopathology

 

 

 

 

 

Slight squamous cell hyperplasia in the forestomach, moderate multifocal necrosis in the liver, slight hepatocellular cytoplasmic alteration (eosinophilic granulation), hypertrophy of the duodenal mucosa, multifocal acute bronchiolitis in the lung and adrenal cortical hypertrophy

m: male

f:: female

PD: premature death

PS: premature sacrifice

TD: test day

For the following tables, please refer to the attached pdf documents under 'Attached background material':

Table 2: Clinical Signs - Summary and individual data

Table 3: Detailed Clinical Observations - Summary and individual data

Table 4: Functional Observations - Summary and individual data

Table 5: Grip Strength - Summary and individual data

Table 6: Spontaneous Motility - Summary and individual data

Table 7: Body Weight - Summary and individual data

Table 8: Body Weight Gain - Summary and individual data

Table 9: Body Weights at Autopsy

Table 10: Absolute Food Consumption - Summary and individual data

Table 11: Relative Food Consumption - Summary and individual data

Table 12: Haematological Parameters - Summary and individual data

Table 13: Biochemical Parameters - Summary and individual data

Table 14: Thyroid Hormone Levels - Summary and individual data

Table 15: Urinalysis - Summary and individual data

Table 16: Ophthalmological and auditory examinations

Table 17: Macroscopic Post Mortem Findings

Table 18: Relative Organ Weights - Summary and individual data

Table 19: Absolute Organ Weights - Summary and individual data

Table 20: Oestrus Cycle

Applicant's summary and conclusion

Conclusions:
The subchronic (90-day) repeated dose toxicity after oral administration by gavage was investigated according to OECD guideline 408 under GLP conditions. Based on the results, the following effect levels were determined: Systemic No-Observed-Adverse-Effect-Level (NOAEL) = 300 mg/kg bw/day and local Lowest-Observed-Adverse-Effect-Level (LOAEL) = 100 mg/kg bw/day.