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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:
15 Sep, 2005 to 20 Jan, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD408, GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on 21 September 1998
Deviations:
yes
Remarks:
Only deviations which were not considered to have compromised the purpose or integrity of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): N-3-Dimethylaminopropyl methacrylamide
- Supplier: Evonik Röhm GmbH, D-64293 Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Stability under test conditions: Stability in water: > 160 hours in water; pure: stable for 3 month
- Storage condition of test material: +2-8 °C, light protected

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: (P) Males/females: approximately 47-49 days old
- Weight at study acclimatisation: (P) 96 - 110 g (male and female), therefore slightly outside the range indicated in the study protocol
- Fasting period before study:
- Housing: No more than 5 per cage of one sex in clear polycarbonate cages measuring 59X38.5X20 cm with a stainless steel
mesh lid and floor (Code 1354 G, Techniplast - Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was
inspected and changed at least 3 times a week.
- Diet: ad libitum, commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy), except as indicated during week 13 of treatment, samples of blood were taken under conditions of food deprivation.
- Water: ad libitum, supplied via water bottles, except as indicated during week 13 of treatment, samples of blood were taken under conditions of
water deprivation.
- Acclimation period: 20 days


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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in distilled water to give the required concentrations of 7.5, 15.0 and 30.0 mg/ml.

The test item was administered orally by gavage at a dose volume of 10 ml/kg body weight.
Control animals received the vehicle alone at the same dose volume.
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for
each animal.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment the proposed formulation procedure was checked by chemical analysis to confirm that the method was
acceptable. Stability over a 24 hour period at room temperature was assessed for content check prior to the start of treatment. Samples of the
formulations prepared at weeks 1 and 13 were analysed to check the concentration. Results of all the analyses were within the limits of acceptance
(95-105%).
Duration of treatment / exposure:
for a minimum of 13 consecutive weeks
All animals were dosed up until the day before necropsy.
Frequency of treatment:
daily, 7 days a week
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle alone), 75, 150 and 300 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
10 male and 10 female per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 75, 150 and 300 mg/kg/day were defined based on information from previous studies (see chapter 7.8.1, Evonik Röhm study, 2002).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (Mortality)
- Time schedule: daily
All observations were recorded for individual animals. Examination of individual animals for signs of reaction to treatment was carried out daily
prior to dosing, immediately after and approximately 1 and 2 hours after dosing up to Day 7 of the study. Since no animals showed any post-dose
effects, examinations were reduced to pre-dose, immediately after and approximately 1 hour after dosing until the end of treatment.

DETAILED CLINICAL OBSERVATIONS AND NEUROTOXICITY: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter each animal was subjected to a detailed clinical examination,
which included an evaluation of neurotoxicity.

All clinical signs 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 (Slightly, Moderately, Severely)
Pronation
Forelimbs drag (Slight, Moderate, Marked)
Hindlimbs drag (Slight, Moderate, Marked)
All observed parameters, with the exception of the pre-dose, are reported in a group incidence table. Individual data are not included in this report.
Once during week 12 an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and assessment of grip strength were also performed.

Motor activity assessment (MA)
The motor activity (MA) of all animals was measured once during week 13 of treatment by an automated activity recording. Measurements were
performed using a computer generated random order.

BODY WEIGHT: Yes
- Time schedule for examinations:
Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to
necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to
necropsy.

FOOD EFFICIENCY:
- 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: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: daily
- Dose groups that were examined: all animals

Both eyes of all animals assigned to the study were examined just prior to the commencement of treatment by means of an ophthalmoscope, and by
a slit-lamp microscope, after the instillation of 0.5% Tropicamide (Visumidriatic®, Visufarma, Rome, Italy). One animal with non-resolving lesions (No. 27580066) was replaced with a spare animal showing no ocular abnormality, from the batch initially ordered for the study. The eyes of all animals
from high dose and control groups were re-examined during week 13 of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13 of treatment
- Anaesthetic used for blood collection: Yes (identity): isofluorane (identity: no data)
- Animals fasted: Yes
- How many animals: all surviving animals from each group to perform coagulation tests. In addition, samples for haematological evaluations were
obtained from 2 female animals (Nos. 27580003 and 27580019), as no suitable samples were collected in the previous bleed.
- Parameters checked: 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

The measurements performed on blood samples are listed below:
-Haematology
Haematocrit
Haemoglobin
Red blood cell count
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
Abnormalities of the blood film
Platelets
Prothrombin time
Partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13 of treatment
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: Clinical chemistry
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: During week 13 of treatment overnight (approximately 16 hours) urine samples were collected from the
animals under the same conditions. Before starting urine collection water bottles were removed from each cage and each animal received
approximately 10 ml/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked:
- Urinalysis
Appearance
Volume
Specific gravity
pH
Protein
Total reducing substances
Glucose
Ketones
Bilirubin
Urobilinogen
Blood
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:
Epithelial cells
Polymorphonuclear leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components

Necropsy
The clinical history of the animal 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 and the required tissue samples preserved in fixative and processed for
histopathological examination (see sections organ weights to Histopathological examination).

Organ weights
From all animals completing the scheduled test period, the organs indicated in the table1 below were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in table 1 below were fixed and preserved in 10% buffered formol saline (except eyes which were fixed in Davidson's
fluid; and testes and epididymides which were fixed in Bouin's solution and all preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for the histopathological examination are listed in table 1 below. After dehydration and embedding in paraffin wax, sections of
the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
The examination was as detailed below:
a) Tissues specified in table 1 below from all animals in the control and high dose groups.
b) All abnormalities in all groups.
In addition, the testes and epididymides of all male animals of each group were dehydrated and embedded in paraffin wax. Sections of the tissues
were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium
(staging of the spermatogenic cycle) was performed on the control and high dose animals.

Table1: of study protocol
---------------------------

Organs / Tissues Weight Fixation Microscopic
Preservation Examination
------------------------------------------------------------------------------------------------------
Abnormalities x x
Adrenal glands x x x
Aorta x x
Bone marrow (from sternum) x x
Brain x x x
Caecum x x
Colon x x
Duodenum x x
Epididymides x x x
Eyes ! x *
Femur with joint x *
Heart x x x
Ileum (including Peyer’s patches) x x
Jejunum x x
Kidneys x x x
Liver x x x
Lungs (including mainstem bronchi) x x
Lymph nodes - cervical x x
Lymph nodes - mesenteric x x
Mammary area x x
Oesophagus x x
Ovaries x x x
Oviducts (a) x x
Pancreas x x
Parathyroid glands (b) x x
Pituitary gland x x
Prostate gland x x
Rectum x x
Salivary glands x x
Sciatic nerve x x
Seminal vesicles x *
Skeletal muscle x *
Skin x x
Spinal column x *
Spinal cord x x
Spleen x x x
Stomach x x
Testes x x x
Thymus x x x
Thyroid x x
Trachea x x
Urinary bladder x x
Uterus - cervix x x x
-----------------------------------------------------------------------------------------------------------
*: not examined as no signs of toxicity or target organ involvement were observed
a: weighed and preserved with ovaries
b: preserved with thyroid gland


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1 section Observation and examinations performed and frequency)
HISTOPATHOLOGY: Yes (see table 1 section Observation and examinations performed and frequency)
Statistics:
For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error variance. The homogeneity of the data was verified by Bartlett's
test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard
deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathology
findings were carried out by means of the non-parametric Kolmogorov-Smirnov test.

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:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study.

No reaction to treatment was seen at daily post-dose examinations. Detailed clinical signs with neurotoxicity assessment did not show any signs
which could be correlated to the treatment with the test item.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related changes were observed in body weights.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant changes were observed in food consumption.

FOOD EFFICIENCY
No data

OPHTHALMOSCOPIC EXAMINATION
No findings were seen at the ophthalmic examination.

HAEMATOLOGY
No changes of toxicological significance were observed in haematological parameters.

CLINICAL CHEMISTRY
A slight but statistically significant increase of cholesterol was observed in animals treated
with 300 mg/kg/day (20% and 22% above controls in males and females, respectively).
However, given the magnitude of this change, it cannot be considered of toxicological importance.
The other statistically significantly variations observed (creatinine, albumin, chloride and sodium) were considered to be incidental.

URINALYSIS
A number of animals treated with 150 and/or 300 mg/kg/day showed a slight increase of urine specific gravity. This increase was statistically
significant in the high dose males and in the mid- and high dose females. A slight reduction of urine volume was also observed in the
mid- and high dose females. Since no other related alterations were observed, no clear conclusion could be made for these alterations and they
cannot be conclusively attributed to treatment.

NEUROBEHAVIOUR
Neurotoxicity tests and motor activity measurements taken at the end of treatment did not show changes clearly attributable to the test item.

ORGAN WEIGHTS
Very slight but statistically significant increases of the absolute and relative weights of the liver (10% greater than controls) were observed in the
males dosed at 300 mg/kg/day. The absolute and relative weights of the kidneys were also increased in the females dosed at 150 and 300 mg/kg/day (7% and 11% the absolute 10% and 13% the relative). These changes were very slight, limited to one sex and not supported by macroscopic and
microscopic examination. Therefore, they were considered of no toxicological importance.

MACROSCOPIC OBSERVATION
No macroscopic finding was described, that could be considered correlated with the administration of the test item.

MICROSCOPIC OBSERVATION
A microscopic examination was performed on all animals in control and high dose groups and on all abnormalities in the remaining animals.
The morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle as described by Leblond & Clermont, 1952) was
performed in the control and high dose males.
No change was observed in the examined tissues/organs, which could be considered treatment-related.
The findings reported were seen to be either expression of spontaneous pathology, commonly seen in this species under our
experimental conditions or incidental in origin. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle
and the integrity of the various cell types present within the different stages. No abnormalities were noted.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No signs of an adverse effect of the test item were seen at any of the dose levels. NOAEL = highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of these results, no signs of an adverse effect of the test item were seen at any of the dose levels investigated (75, 150 and 300 mg/kg/
day). Therefore, the high dose of 300 mg/kg/day, when administered daily for 13 consecutive weeks, was considered the No Observed Adverse
Effect.
Level (NOAEL).
Executive summary:

The oral toxicity of N-3-DIMETHYLAMINOPROPYLMETHACRYLAMIDE (CAS 5205- 93-6) when given by daily administration to rats, has been investigated over a period of 13 consecutive weeks. Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by gavage at dosages of 75, 150 and 300 mg/kg/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (distilled water) and acted as a control.

No signs of toxicity were observed at post-dose examinations or at the weekly clinical examination. No changes were observed in body weights or food consumption. Some slight, statistically significant changes were sporadically observed in clinical chemistry

parameters (increase of cholesterol) and urinalysis (increment of specific gravity) in animals dosed at 300 and occasionally, at 150 mg/kg/day. These changes were not consistent between sexes, insufficient in magnitude and, therefore, considered of no toxicological

importance.

No treatment-related abnormalities were observed at post mortem examination. The slight statistically significant increases of liver and kidneys weights, observed in the males dosed at 300 mg/kg/day and in the females dosed at 150 and 300 mg/kg/day, respectively, were

limited to one sex and not supported by macroscopic and microscopic examination. Therefore, they were considered of no toxicological importance.

Histopathological examination did not reveal any treatment-related changes.

On the basis of these results, no signs of an adverse effect of the test item were seen at any of the dose levels investigated (75, 150 and 300 mg/kg/day).

Therefore, the high dose of 300 mg/kg/day, when administered daily for 13 consecutive weeks, was considered the No Observed Adverse Effect Level (NOAEL).

This study is acceptable and satisfies the guideline requirement for a 13 week oral toxicity study (OECD 408) in rats.

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