3-((C12-18)-acylamino)-N-(2-((2-hydroxyethyl)amino)-2-oxoethyl)-N,N-dimethyl-1-propanaminium chloride

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

3.1 AnimaIs and Animal Husbandry
A sufficient number of male and female Sprague-Dawley Crl:CD®BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. On receipt the animals were examined for signs of ill-health or injury. The animaIs were acclimatised for nine days during which time their health status was assessed.
A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 174 to 212g, and the females weighed 147 to 203g, and were approximateiy six to seven weeks old.
The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water. A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) was used. Certificates of analysis of the batches of diet are given in Appendix XVII. Mains water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in an air-conditioned room within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidities were included in the study records. The temperature and relative humidity were controlled to remain within target ranges of 21 ± 2C and 55 ± 15% respectively. Occasional deviations from the relative humidity target were considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to treatment groups using random letter tables, and the group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steei cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg/day of distilled water.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
1.5 mg/ kg bw/day
Basis:
other: calculated from bodyweight

Remarks:

Doses / Concentrations:
15 mg/kg bw/day
Basis:
other: calculated from bodyweight

Remarks:

Doses / Concentrations:
150 mg/ kg bw/day
Basis:
other: calculated from bodyweight

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends. All observations were recorded.

Functional Observations
Prior to the start of treatment and on Days 2, 9, 15 and 22 all animals were observed for signs of functional/behavioural toxicity. On Day 22 functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors / Skin colour
Twitches / Respiration
Convulsions / Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour /Urination
Salivation / Defecation
Pilo-erection / Transfer arousal
Exophthalmia / Tail elevation
Lachrymation

Functional Performance Tests
a) Motor Activity
Twenty purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested on either occasion and were randomly allocated to the activity monitors.
For each test, the animals were allowed ten minutes equilibration time. The tests were performed at approximateiy the same time of the day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final six minute period (considered to be the asymptotic period).
b) Forelimb/Hindlimb Grip Strength
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip of each animal was made. Two consecutive trials were performed for each animal.

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The scoring system used is outlined in Appendix I. The foilowing parameters were observed:
Grasp response / Touch escape
Vocalisation / Pupil reflex
Toe pinch / Startle refiex*
Tail pinch / Blink reflex
Finger approach
* Startle reflex was measured using the ST1058 Startle Test Meter (Benwick Electronics). Each animal was placed on the force platform and allowed to settle. An audible tone was activated and any change in the force exerted by the animal on the force platform, as a result of startle induced tremor, was measured. The percentage average response, root of the mean square and peak response were calculated for each animal and two consecutive trials were performed.

Bodyweight
Individuai bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

Food Corisumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animais from each test and control group at the end of the study (Day 28). Blood samples were obtained from the laterai tail vein. Animals were not fasted prior to sampling.
The methods used for haematological and biood chemical investigations are given in Appendix XIX and normal ranges are shown in Appendix XXI.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Cresyl blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Hepato Quick’ and Activated partial thromboplastin time (APTT) was assessed by ‘Preci Clot’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Calcium (Ca+ +)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chioride (CI-)

Sacrifice and pathology:

On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone (Sagatal, 60 mg/mL; May and Baker
Limited, Dagenham, Essex, UK) followed by exsanguination.
All animals were subjected to a full external and internai examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
- Adrenals Brain Epididymides Heart
- Kidneys Liver Ovaries Spleen
- Testes Thymus

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: Adrenals Muscle (skeletal), Aorta (thoracic) Oesophagus, Bone & bone marrow (femur including stifle joint), Ovaries, Pancreas, Bone & bone marrow (sternum) Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Colon, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum, jejunum, Kidneys, Liver, Iungs (with bronchi) #, Lymph nodes (cervical and mesenteric), Rectum, Sahvary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical), Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Trachea, Urinary bladder, Uterus
# Lungs were inflated to approximately normal inspiratory volume with buffered lO% formalin before immersion in fixative

Microscopic examination was conducted by the Study Pathologist. Ail findings were entered into the ROELEE 84 pathology computerisation system for tabulation and report production.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous pairwise comparisons were conducted using Dunnetts’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and MannWhitney “U test.
Following data analysis of plasma bilirubin concentration among control and test animaIs, statistical significance was assigned to females from the 15 mg/kgldy treatment group in comparison with controls. The difference in mean and standard deviation between these two groups was negligible and the results of this analysis were rejected for contravening the function of Dunnett’s pairwise comparison test. Under these conditions, single t-tests were performed to compare control values with those of treatment groups where an intergroup difference was apparent.
The haematology variable basophils was not analysed since consistently greater than 3O% of the data were recorded as the same value.
Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p 0.05 (not significant)

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 examined

Water consumption and compound intake (if drinking water study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioural Assessments No treatment-related changes were detected. Functional Performance Tests No treatment-related changes were detected. Sensory Reactivity Assessments No treatment-related changes were detected.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality
There were no deaths during the study.
Clinical Observations
No clinically observable signs of toxicity were detected during the study.
Functional Observations
Behavioural Assessments
No treatment-related changes were detected.
Functional Performance Tests
No treatment-related changes were detected.
Sensory Reactivity Assessments
No treatment-related changes were detected.
Bodyweight
No adverse effect on bodyweight development was detected.
Food Consumption
No adverse effects on dietary intake were detected.
Water Consumption
No intergroup differences were detected.
Haematology
No treatment-related changes were detected.
Blood Chemistry
No treatment-related changes were detected.
Organ Weights
No treatment-related changes were detected.
Necropsy
No treatment-related macroscopic abnormalities were detected.
Histopathology
No treatment-related microscopic abnormalities were observed.

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

Repeated oral administration of the test material to rats by gavage for a period of twenty-eight consecutive days at dose levels of up to 150 mg/kg/day produced no treatment-related changes in the parameters measured. The ‘No Observed Effect Level’ (NOEL) was, therefore, considered to be 150 mg/kg/day.

Executive summary:

The study was designed to investigate the systemic toxicity of the test material.

It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 “Repeated Dose 28 Day Oral Toxicity Study in Rodents” (Adopted 27 JuIy 1 995).

 

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD®BR strain rats, for twenty-eight consecutive days, at dose levels of 1.5, 15 and 150 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (distilled water).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaiuated for all animals at the end of the study. Ail animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

 

The results are summarised as follows:

Mortality

There were no deaths during the study.

Clinical Observations

No clinically observable signs of toxicity were detected during the study.

Functional Observations

Behavioural Assessments

No treatment-related changes were detected.

Functional Performance Tests

No treatment-related changes were detected.

Sensory Reactivity Assessments

No treatment-related changes were detected.

Bodyweight

No adverse effect on bodyweight development was detected.

Food Consumption

No adverse effects on dietary intake were detected.

Water Consumption

No intergroup differences were detected.

Haematology

No treatment-related changes were detected.

Blood Chemistry

No treatment-related changes were detected.

Organ Weights

No treatment-related changes were detected.

Necropsy

No treatment-related macroscopic abnormalities were detected.

Histopathology

No treatment-related microscopic abnormalities were observed.

 

Conclusion:

Repeated oral administration of the test material to rats by gavage for a period of twenty-eight consecutive days at dose levels of up to 150 mg/kg/day produced no treatment-related changes in the parameters measured. The ‘No Observed Effect Level’ (NOEL) was, therefore, considered to be 150 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat