Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 June 1995 to 6 November 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

EC Number:

421-660-1

EC Name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

Molecular formula:

Hill formula: C50 H96 N2 O10 CAS formula: C42 H74 O8. 2(C4 H11 N O)

IUPAC Name:

bis((2-hydroxyethyl)dimethylazanium) (4E)-3-{[2-({3-carboxylato-3-[(2E)-hexadec-2-en-2-yl]propanoyl}oxy)ethoxy]carbonyl}-4-methyloctadec-4-enoate

Constituent 2

Reference substance name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

IUPAC Name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river (UK) Limited
- Age at study initiation: approximately 5-6 weeks
- Weight at study initiation: 119 - 176 g
- Fasting period before study: None
- Housing: Polypropylene grid-floor cages
- Diet (e.g. ad libitum): free access, exept during urine collection when food was withdrawn overnight
- Water (e.g. ad libitum): free access
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared at the appropriate concentration as a solution in Arachis Oil B.P. Analysis of the stability of these formulations showed them to be stable for at least 10 days. Formulations were therefore prepared weekly and stored at 4 deg C in the dark.


DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): NDA
- Concentration in vehicle: 0 - 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
- Lot/batch no. (if required): NDA
- Purity: NDA

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analysed by HPLC following extraction with methanol to give a final theortical test material concentration of approixmately 1 mg/ml. Results of analysis indicates that the prepared formulations were within +/- 10% of the nominal concentration.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day active ingredient (control)
Basis:
other: nominal via gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg/day active ingredient
Basis:
other: nominal via gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 mg/kg/day active ingredient
Basis:
other: nominal via gavage

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg/day active ingredient
Basis:
other: nominal via gavage

No. of animals per sex per dose:

Male: 10 animals at 0 mg/kg bw/day (5 recovery)
Male: 5 animals at 50 mg/kg bw/day
Male: 10 animals at 150 mg/kg bw/day (5 recovery)
Male: 10 animals at 1000 mg/kg bw/day (5 recovery)
Female: 10 animals at 0 mg/kg bw/day (5 recovery)
Female: 5 animals at 50 mg/kg bw/day
Female: 10 animals at 150 mg/kg bw/day (5 recovery)
Female: 10 animals at 1000 mg/kg bw/day (5 recovery)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels based upno a preliminary range finding study. Animals were dosed at 0, 150, 400 and 1000 mg/kg/day (6 rats per dose level) for 14 days. There were no deaths during this study. 5 of the animals treated at 1000 mg/kg/day showed gross liver changes at necropsy. Animals from the remaining groups showed no macroscopic abnormalities.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): Random

Positive control:

Not used

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, one and five hours after dosing during the week, immediately before dosing and one hour after dosing at weekends.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: As above - examined for overt signs of toxicity, ill-health or behavioural change


BODY WEIGHT: Yes
- Time schedule for examinations: Days 0 (before treatment), 7, 14, 21 and 28. Also on Days 35 and 42 for the recovery groups. Bodyweights also recorded at terminal kill.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): N/A
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data


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


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:


HAEMATOLOGY: Yes
- Time schedule for collection of blood: For all surviving animals at end of treatment period (Day 28) and at end of recovery period (Day 42)
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All surviving animals
- Parameters were examined: Haemoglobin, Erythrocyte count, Haematocrit, Erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin concentration), Total leucocyte count, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils and basophils), Platelet count, Methaemoglobin and Reticulocyte count. Clotting (prothrombin) time was also assessed.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: For all surviving animals at end of treatment period (Day 28) and at end of recovery period (Day 42)
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All surviving animals
- Parameters were examined: Urea, Glucose, Total protein, Albumin, Albumin/Globulin ratio (by calculation), Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Gamma glutamyl transpeptidase, Aspartate aminotransferase, Alkaline phosphatase, Creatinine, Triglycerides, Total cholesterol and Total bilirubin


URINALYSIS: Yes
- Time schedule for collection of urine: All surviving animals from non-recovery groups during Week 4, all recovery group animals during the final week of the study.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters were examined: Volume, Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Reducing substances and Blood.


NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

On completion of the dosing period or, in the case of recovery group animals, at the end of the treatment-free period, all surviving 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 internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:

Adrenals Gonads Kidneys Pituitary
Brain Heart Liver Spleen



HISTOPATHOLOGY: Yes

Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:


Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum) Brain
Caecum
Muscle (skeletal)
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Colon
Duodenum
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver Lungs
Lymph nodes (cervical and mesenteric)
Salivary glands
Sciatic nerve
Seminal vesicles
Skin (hind limb)
Spleen
Stomach
Testes
Thymus Thyroid/parathyroid Trachea
Urinary bladder
Uterus

All tissues were despatched to Finn International, One Eyed Lane, Weybread, Diss, Norfolk, UK for processing. The following preserved tissues from allcontrol and high dose group animals (Groups 1 and 4) were prepared as paraffin blocks, sectioned at nominal thickness of 5 pm and stained with haematoxylin and eosin for subsequent microscopic examination:
Adrenals
Heart
Kidneys
Liver
Spleen
Testes

Macroscopically observed lesions were also processed.

Since there were indications of treatment-related hepatic, renal and splenic changes, examination was subsequently extended to include similarly prepared sections of liver, kidneys and spleen from all animals in the remaining dose groups, including those in the recovery groups.

Other examinations:

None

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.

Absolute and relative organ weights, weekly bodyweight gain, haematological and blood chemical data and quantitative urinalytical data were analysed by one way analysis of variance incorporating either 'F-max' test or Levene's test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal-Wallis non-parametric analysis of variance and Mann Whitney U-Test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical observations:
There were no abnormal clinical observations apparent in the
vehicle control animals.


Clinical signs of toxicity were first observed for the rats
dosed at the highest level during week 1 and included
salivation, hunched posture, diuresis, dehydration, tiptoe
gait, emaciation , noisy respiration, pale yellow
extremities, fur loss, wet fur and red to brown staining on
the fur (around the mouth, nose, eyes and ano-genital
region). One female rat was found dead at 12 days. Recovery
was complete by day 10 of the recovery period. In high dose
animals bodyweight gain was most severely affected during
the first two weeks of treatment with weight gain reduced by
55% and 64% in males and females respectively. There was
recovery on decrease of top dose (see comments) although
body weight gain was still reduced by 36% and 19% in males
and females respectively at 28 days. Recovery group (1000
mg/kg/day) animals of either sex showed similar body weight
gains to controls during the 14 day treatment-free period.

There were no signs of toxicity apparent in any animal in
the 150 or 15 mg/kg/day dose group.

Laboratory findings:
Hematology:

At 1000 mg/kg/day, both sexes had limited (c10%) but
statistically significant decreases in hemoglobin,
erythrocyte count and hematocrit compared to controls.
Statisically significant increase in total leucocyte count
of 38% and 91% in males and females respectively
andreticulocyte count 100% and150% in males and females
respectively were noted. Females also had a statistically
significant increase in mean corpuscular volume (MCV) of
approximately 10%; males exhibited a slight increase in
clotting time of 16%.


No significant changes were noted in animals dosed at 150 or
50 mg/kg/day. Other than the statistically significant
effects seen in the high dose animals, no dose response was
observed in the haematological effects.


Blood Chemistry:

Male and female rats dosed with 1000 mg/kg/day showed a
statistically significant increase in aspartate
aminotransferase (ASAT) of 62 and 95% respectively and
alanine aminotransferase (ALAT) of 150% and 125%
respectively compared to the control animals. When compared
to controls, females at this level (predominantly 2 animals)
showed reductions in plasma total protein, albumin
concentration of 12% and 20% respectively and increases
inalkaline phosphatase (AP), cholesterol and bilirubin
concentrationsof 64%, 55% and 52% repectively. The changes
in the blood parameters which were considered to be
significant toxicologically were reversible after 14 days
post-treatment.


No toxicological significant changes in the blood chemical
parameters were noted for any animal dosed with 150 or 50
mg/kg/day.


Urinalysis:

Diuresis (increase volume of urine of reduced specific
gravity) was apparent in 3 males dosed at 1000 mg/kg/day.
Affected males in the satellite group recovered from this
condition during the 14-day post-dose period.


No toxicologically significant changes in measured urine
parameters were noted in the females dosed at 1000 mg/kg/day
or in any animal in the lower dose groups.

Effects in organs:
Organ Weights:

Males and females dosed at 1000 mg/kg/day had an increased
relative liver weight of 26% and 79% respectively and spleen
weight of 36% and 100% respectively. Females also had
higher absolute liver and spleen weights of 57% and 76%
respectively and males had an increased relative kidney
weightof 22%. Females in the 1000 mg/kg/day satellite group
(14 days without treatment) showed an increased relative
liver and spleen weight and an increased absolute spleen
weight. The male recoveryanimals showed an increased
relative liver weight.


There were no toxicologically significant organ weight
changes observed for any animal in the 150 or 50 mg/kg/day
dose groups.


Necropsy:

At 1000 mg/kg/day, the 4 surviving females and 4/5 males
exhibited macroscopic liver abnormalities including an
accentuated lobular pattern, pallor and/or a granular
appearance. At the end of recovery 1000 mg/kg/day, an
accentuated lobular liver pattern was observed in all 5
females and 1 male. In addition, 2 females had livers with
a granular appearance. The female rat that died during the
study exhibited darkened liver, lungs and kidneys.


No toxicologically significant macroscopic abnormalities
were noted in animals receiving 150 or 50 mg/kg/day.


Histopathology:

In the liver, incidences of bile duct proliferation,
periportal stromal fibrosis and periportal accumulations of
pigment were evident in animals dosed with 1000 mg/kg/day.
In the recovery group at this level, most of the conditions
partially regressed; periportal pigment deposits did not.
One female at 150 mg/kg/day had bile duct proliferation.


In the kidneys, animals dosed with 1000 mg/kg/day had
pigment accumulations in the tubular epithelium. This
condition completely regressed in male recovery group rats
and partially regressed in females.


In the spleen, granular pigment deposits were apparent in
males and females at 1000 mg/kg/day, but didn't regress in
the recovery group.


No significant microscopic effects were noted in males at
150 mg/kg/day or in any animal at 50 mg/kg/day.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Classified as: Not classified

Oral administration of the test material, OS 114451A, to rats, by gavage, at dose levels of 50, 150 and 1000 (then 500) mg/kg/day produced a number of toxicologically significant changes at a dose level of 1000 mg/kg/day and minimal bile duct proliferation for one female dosed at 150 mg/kg/day. Most of the treatment-related effects were reversible after an additional fourteen days without treatment although several toxicologically significant changes, especially those involving the liver, were still present at the end of the recovery period amongst animals dosed at 1000 mg/kg/day. No toxicologically significant changes were observed amongst males dosed at 150 mg/kg/day or amongst animals of either sex dosed at 50 mg/kg/day and the 'No Observed Effect Level' (NOEL) was therefore considered to be 150 mg/kg/day for males and 50 mg/kg/day for females.

The isolated treatment-related change observed at a dose level of 150 mg/kg/day was considered not to be indicative of serious damage to the health of the animals, as defined by the criteria given in the EC labelling guide of Directive 93/21/EEC.

Executive summary:

Oral administration of the test material,OS114451A,to rats, by gavage, at dose levels of50,150and 1000 (then500)mg/kg/day produced a number of toxicologically significant changes at a dose level of 1000 mg/kg/day and minimal bile duct proliferation for one female dosed at 150 mg/kg/day. Most of the treatment-related effects were reversible after an additionalfourteen days without treatment although several toxicologically significant changes, especiallythose involving the liver, were still present at the end of the recovery period amongst animals

dosed at 1000 mg/kg/day. No toxicologically significant changes were observed amongst males dosed at 150 mg/kg/day or amongst animals of either sex dosed at 50 mg/kg/day and the 'No Observed Effect Level' (NOEL) was therefore considered to be 150 mg/kg/day for males and 50 mg/kg/day for females.

 

The isolated treatment-related change observed at a dose level of 150 mg/kg/day was considered not to be indicative of serious damage to the health of the animals, as defined by the criteria given in the EC labelling guide of Directive 93/21/EEC.