Registration Dossier

Administrative data

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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Remarks:
statement
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch: 19902-1
Expiry date: 31 October 2006

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 * 3.0•‹C (actual range: 20.4- 22.1 OC), a relative humidity of 30-70% (actual range: 36 - 87%) and 12 hours artificial
fluorescent light and 12 hours darkness per day.
Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Temporary fluctuations from the liahtldark cycle (with a maximum of 1 hour) occurred due toperfo&anc6 of functional observati6ns in the
room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation
Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type Ill, height 15 cm.) with sterilised sawdust (Woody-Clean type 314, Tecnilab-BMI BV, Someren, The Netherlands) provided as bedding. Results of bedding analyses for contaminants are examined and archived.
Diet
Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Water
Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Results of analysis for ingredients andlor contaminants of diet, sawdust and water were assessed and did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 423798.
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1000 mg/kg/day recovery 14 days
Basis:
nominal in water
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
other: concurrent vehicle with recovery 14 days
Details on study design:
- Dose selection rationale: Based on the results of a 5-day range finding study
- Post-exposure recovery period in satellite groups: 14 days after discontinuation of dosing protocol

Examinations

Observations and examinations performed and frequency:
Mortality / Viability:
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENVIJMIMONOI 200017). The time of death was recorded as precisely as possible.
Clinical signs:
At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate,grade 3 = severe, grade 4 = very severe
Functional Observations
During week 4 of treatment, the following tests were performed on allanimals (abbreviations mentioned in the respective tables indicated
between brackets):
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static
righting reflex (STATIC R) and grip strength (GRIP) (Score 0 =
normall present, score 1 = abnormallabsent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Since treatment-related effects during motor activity tests were seen in week 4, these tests were repeated in week 6.
Ophthalmoscopic examinations:
Both eyes were examined following instillation of tropicamide solution (5 mglml): week 4 (all animals) Since no treatment-related ophthalmoscopic effects were noted in
week 4, no ophthalmoscopic examination was performed in week 6
Body weights:
Treatment period: on days 1, 8. 15, 22 and 28.
Recovery period: on days 1,8 and 14.
Food consumption:
Weekly
Water consumption:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical Laboratory Investigations
At end of treatment and after recovery, blood samples were collected under iso-flurane anaesthesia between 7.30 and 9.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all ratslsexlgroup and collected into tubes prepared with EDTA for haematological parameters (0.5 mi), with citrate for clotting tests (1.0 ml) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 ml).
The following parameters were determined
Haematology
Erythrocytes count
Haemoglobin
Haematocrit
Mean corpuscular volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
Platelet count
Red cell distribution width
Total leucocytes count
Differential leucocyte count (neutrophils (NEUT), lymphacytes (LYMPHO), monocytes (MONO),eosinophils (EQS), basophils (BASO)

Clotting Potential - Prothrombin time PT-Partial thromboplastin time APlT

Clinical Biochemistry
Alanine aminotransferase
Alkaline phosphatase
Aspartate aminotransferase
Bilirubin, total
Chloride
Cholesterol, total
Creatinine
Glucose
Phosphorus INORG.PHOS
Protein, total
Protein, albumin
Urea
Calcium
Potassium
Sodium
Sacrifice and pathology:
All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the
following tissues and organs were collected from all animals found dead and at planned necropsy and fixed in a neutral phosphate buffered 4%
formaldehyde solution:
Identification marks: not processed
Adrenal glands
Aorta
Brain (cerebellum, mid-brain, cortex)
Caecum
Cervix
(Clitoral gland)
Colan
Duodenum
Epididymides
(Eyes with optic nerve and Harderian gland)
(Female mammary gland area)
(Femur including joint)
Heart
Ileum
Jejunum
Kidneys
(Larynx)
(Lacrimal gland, exorbital)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Oesophagus
Ovaries
Pancreas
Peyer's patches (jejunum, ileum) if detectable
Pituitary gland
(Preputial gland)
Prostate gland
Rectum
(Salivary glands - mandibular, sublingual)
Sciatic nerve
(Seminal vesicles)
(Skeletal muscle)
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid including parathyroid
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions
Other examinations:
The following organ weights (and terminal body weight) were recorded from the surviving
animals on the scheduled day of necropsy:
Adrenal glands Brain
Epididymides Heart
Kidneys Liver
Ovaries Spleen
Testes Thymus
Statistics:
Analysis of variants with multiple comparison procedures according DUNNETT. p<0.05

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All rats at 0, 50 or 150 mglkg/day survived the scheduled duration of the study. At 1000 mglkglday, 3 males and 7 females died or were killed in a moribund state in the course of the treatment phase.
Mortality:
mortality observed, treatment-related
Description (incidence):
All rats at 0, 50 or 150 mglkg/day survived the scheduled duration of the study. At 1000 mglkglday, 3 males and 7 females died or were killed in a moribund state in the course of the treatment phase.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains at 50 or 150 mg/kg/day were similar to those of control animals. The surviving males and females at 1000 mg/kg/day showed a slightly reduced weight gains relative to controls by the end of the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 50 or 150 mg/kg/day - similar to control animals. At 1000 mg/kg/da - the animals ate less food in treatment weeks 1 and 4, females more affected.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects in week 4, no need to examine in week 6
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Lower proportion total lymphocyte counts, associated with a higher proportion of neutrophils and monocytes; band neutrophils were detected in one male. No such deviations were seen at the end of the recovery period, so the effects are probably reversible.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Lower levels of glucose in males at 1000 mg/kg/day, and lower levels of total protein and albumin in males and females at 1000 mg/kg/day, and lower total protein levels in females at 150 and 50 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/b.w.-lower absolute epididymides weights and higher relative brain, liver, kidneys and testes weights in males, and higher relative spleen and adrenal weights in females after treatment or recovery, respectively.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related gross findings in the stomach at 1000 mg/kg/day
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Centrilobular macrovesicular vacuolation and microgranuloma centrilobular macrovesicular vacuolation and microgranuloma found in the liver at 1000 mg/kg/day.
Histopathological findings: neoplastic:
not examined
Details on results:
Clinical signs observed in animals at 1000 mglkglday:
All males and females showed a brown discolouration of the urine by the end of week 1 up to the end of the treatment phase
Diarrhoea was observed in 2 males and 3 females in the first week of treatment
Salivation was noted in 5 males and 5 females during the treatment phase
Squeaking was observed in 4 males and piloerection andlor a hunched posture was observed in 4 females, many of which subsequently died or were killed in a moribund state.
Squeaking, rales and ptosis in males, and lethargy, tremor, gasping, pale appearance, ptosis, hunched posture and swelling of the abdomen in females were noted prior to unscheduled.
Functional Observations: Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. Statistically significant differences were found for high dose males during motor activity assessment with the lower sensors. Motor activity assessment with animals after the recovery period did not indicate any difference with control animals, albeit that only one high dose female and 3 high dose males survived to the end of recovery.
Clinical biochemistry :Lower total protein and albumin levels were not reversed completely at the end of the recovery period in high dose males, suggesting that these changes are persistent.

Effect levels

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on histopathology results in the liver at 1000 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for N-{2-[2-(DIMETHYLAMINE)ETHOXv]ETHYL}-N-METHYL-l,3-PROPANEDlAMlNE of 150 mg/kg/day was established.
Executive summary:

Following the administration of N-{2-[2-(DIMETHYLAfvlINE)ETHOXY]ETHYL)-N-METHYL-1,3-PROPANEDIAMINE by once daily gavage to Wistar rats at doses up to 1000 mg/kg for twenty eight days, there was mortality and morphologic alterations in the stomach, liver and spleen at 1000 mglkglday, and no toxicity 150 and 50 mgkg/day.

Ten animals at 1000 mg/kg/day died or were killed in a moribund state in weeks 1 and 4. In two animals there was evidence of misgavage, in another animal inanition was considered to have contributed to death of moribundity.

Findings observed in the motor activity assessment of males dosed at 1000 mg/kg/day at least suggest that they were more stationary in the cages and may be related to the discomfort observed during clinical observations. Although this finding is related to treatment with the test substance, there are no corroborative histopathological findings or findings at clinical pathology that suggest adverse effects of neural dysfunction on motor activity caused by the test substance. In the high dose, only one female and three males survived to the recovery period. Taking into account these small groups sizes, there seemed to be no difference after recovery in the motor activity of treated animals compared to controls. Macroscopic observations at 1000 mg/kg/day were thickened limiting ridge in the stomach and an irregular surface in the forestomach. Together with the observed microscopy of the stomach (glandular epithelial erosion, forestomach squamous hyperplasia, ulceration of the forestomach and glandular inflammation) these findings are considered to be caused by local irritation reactivity of the test substance and are therefore considered being unrelated to systemic toxicity. Histopathological findings that were considered to be systemic were found in the liver at 1000 mg/kg/day and consisted of centrilobular macrovesicular vacuolation and microgranuloma. The observed shift in type of white blood cells (reduced lymphocytes with neutrophilia) might be a secondary non-specific response to stress associated with treatment. Therefore, the change in white blood cells may be considered not to be of primary toxicological significance.

The persisting lower total protein and albumin levels in the recovery animals suggest that there is an irreversible alteration in the protein synthesis in these animals. Similar changes in the animals dosed at 50 and 150 mg/kg/day were not corroborated by any other findings that suggest adversely altered protein metabolism or related liver dysfunction and is therefore considered to be of no toxicological importance. From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for N42-[2-(DIMETHYL4MINE)ETHOXYlETHYL)-N-METHYL-I ,3-PROPANEDlAMlNE of 150 mglkglday was established.