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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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: GLP study, OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsors identification : LIPACIDE UG
Description : off white solid
Batchnumber : 00 171 300
Date received : 01 August 2000
Storage conditions : room temperature, in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD® (SD) IGS 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 animals were acclimatised for eleven 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 164 to 212g, and the females weighed 150 to 175g 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. A certificate of analysis of the batch of diet used is given in Appendix 13. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered flot to contain any contaminant at a level that might have affected the purpose of integrity of the study. Environmental enrichment was provided in the form ofwooden chew blocks (B & K Universal Ltd., Huli, UK).
The animals were housed in a single 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 tweive hours darkness. Environmental conditions were continuousiy 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 ± 2°C and 55 ± 15% respectively.
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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The test material was administered daily, for up to twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposabie plastic syringe. Control animais were treated in an identicai manner with 4 ml/kg/day ofArachis ou BP.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervais.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
For the purpose of this study the test material was prepared at the appropriate concentrations as a suspension in Arachis ou BP. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark.
Samples were taken of each test material formulation and were anaiysed for concentration of Lipacide UG at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within ± 8% ofthe nominal concentration.

Examinations

Observations and examinations performed and frequency:
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 wherever possible. Animals were observed immediately before dosing and one hour after dosing at weekends.

Prior to the start of treatment and on Days 7, 14, 21 and 24 ail surviving animals were observed for signs of functional/behaviourai toxicity. Functional performance tests were also performed on all surviving animals during Week 4 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
Detalled 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 Paipebral ciosure
Bizarre/Abnormal/Stereotypic behaviour Urination
Salivation Defecation
Piio-erection Transfer arousal
Exophthalmia Tau elevation
Lachrymation

Functiona! Performance Tests
Motor Activity.
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overail sixteen hour period and also during the final 20% of the period (considered to be the asymptotic period).
Forelimb/Hindljmb Grip Strength.
An automated grip strength meter was used. Each animal was allowed to grip the proxirnal metal bar of the meter with its forepaws. The animal was pulled by the base of the tau until its grip was broken. The animal was drawn along the trough of the meter by the tail untii its hind paws gripped the distal metal bar. The animal was puiled by the base of the tau until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity
Each animal was individualiy assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Startie reflex
Tau pinch Blink reflex
Finger approach

Bodyweight
Individual bodyweights were recorded on Day O (the day before the start of treatment) and on Days 7, 14, 21 and 28 wherever possible. Bodyweights were also recorded at terminal kill.
Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.
Water Consumption
Water intake was observed daiiy, for each cage group, by visual inspection of the water botties for any overt changes.

Laboratory Investigations
Haematoiogical and blood chemical investigations were performed on ail surviving animais from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampiing.
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 siides 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 dot’ using samples cBlood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Calcium (Ca++)
Glucose Inorganic phosphorus (P)
Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT)
Albumin Alanine aminotransferase (ALAT)
AlbuminlGlobulin (A/G) ratio (by calculation) Alkaline phosphatase (AP)
Sodium (Na+) Creatinine (Creat)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)
Sacrifice and pathology:
Pathology
On compietion of the dosing period ail surviving animais were killed by intravenous overdose of sodium pentobarbitone foiiowed by exsanguination.
Ail animais were subjected to a fuli extemal and internai examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animais that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spieen
Heart Testes
Kidneys Thymus
Normal ranges for these organ weights are given in Appendix 18.

Histopathology
Samples of the following tissues were removed from ail animais and preserved in buffered 10% formalin:
Adrenals Oesophagus
Aorta (thoracic) Ovaries
Bone & bone marrow (femur inciuding stifle joint) Pancreas
Boue & boue marrow (sternum) Pituitary
Brain (including cerebrum, cerebellum and pons) Prostate
Caecum Rectum
Colon Saiivary glands (submaxiiiary)
Duodenum Sciatic nerve
Epididymides Seminal vesicles
Eyes Skin (hind limb)
Gross lesions Spinal cord (cervical)
Heart Spleen
Ileum Stomach
Jejunum Testes
Kidneys Thymus
Liver Thyroid/parathyroid
Lungs (with bronchi) # Trachea
Lymph nodes (cervical and mesenteric) Urinary bladder
Muscle (skeletal) Uterus

All tissues were despatched to Propath UK Ltd, Wiilow Court, Netherwood Road, Rotherwas, Hereford, UK for processing. The tissues shown in bold from ail control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 jim and stained with haematoxylin and eosin for subsequent microscopic examination. The liver and spleen from ail 150 and 15 mg/kg/day dose group animals were also processed.
Since there were indications of treatment-related changes in the stomach, trachea and lungs examination was subsequently extended to include simiiarly prepared sections of these tissues from ail animais in the other treatment groups.
Microscopic examination was conducted by the Study Pathologist. Ail findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Haematological, biood chemical, organ weight (absolute and relative to terminal bodyweight), weekiy bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis foilowed 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 Dunnett’s test. Where Levene’ s test showed unequal variances the data were anaiysed using non-parametric methods: Kruskal-Waliis ANOVA and Mann-Whitney ‘U’ test.
The haematology variable basophiis was flot analysed since consistentiy greater than 30% of the data were recorded as the same value.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One female treated with 1000 mg/kg/day was found dead at the start of Day 24. There were no other unscheduled deaths during the study period.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female treated with 1000 mg/kg/day was found dead at the start of Day 24. There were no other unscheduled deaths during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No abnormal bodyweight changes were detected for 150 or 15 mg/kg/day animais.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No such effects were detected for 150 or 15 mg/kg/day animais.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water botties revealed no intergroup differences.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No such effects were detected for 1000 mgi’kg/day females or for animais of either sex treated with 150 or 15 mg/kg/day.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related macroscopic abnormalities were detected at 150 or 15 mg/kg/day.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
Mortality
One female treated with 1000 mg/kg/day was found dead at the start of Day 24.
There were no other unscheduled deaths during the study period.

Clinical Observations
Males and females treated with 1000 mg/kg/day showed increased salivation either prior to or up to ten minutes after dosing from Day 3 onwards, occasionally persisting up to an hour after dosing as the study progressed. Noisy respiration and hunched posture developed from Days 7 or 10 respectively and increased in incidence throughout the treatment period. On the final day of treatment four animais showed gasping and/or laboured respiration. Other findings included tiptoe gait noted in both sexes during the final week of treatment, pilo-erection in one female on Days 10 and 11 and incidents of wet and stained fur and staining around the mouth. Animais of either sex treated with 150 mg/kg/day showed increased salivation up to ten minutes after dosing during the final week of treatment accompanied by hunched posture at this time in females only.
No abnormalities were detected in animais treated with 15 mg/kg/day.

Bodyweight
Reduced bodyweight gain was detected for animals of either sex treated with 1000 mg/kg/day during Weeks 3 and 4 compared to controls achieving statistical significance for males (p<0.01) on both occasions. During the final week of the study two males and one female showed a bodyweight loss.
No abnormal bodyweight changes were detected for 150 or 15 mg/kg/day animais.


Food Consumption
Reduced dietary intake was detected for 1000 mg/kg/day animals during Weeks 3 and 4 compared to controis, the effect more severe in males than femaies. Food efficiency (the ratio of bodyweight gain to food consumption) was also reduced during this period.
No such effects were detected for 150 or 15 mg/kg/day animais.


Blood Chemistry
Males treated with 1000 mg/kg/day showed a statistically significant reduction in total plasma protein (p<0.0l) compared with controls with two individuai values outside the normal range for rats of the strain and age used. Plasma albumin was also reduced but statistical significance was flot achieved. Plasma cholesterol was reduced (pNo such effects were detected for 1000 mg/kg/day females or for animais of either sex treated with 150 or 15 mg/kg/day.

Necropsy
A summary incidence of necropsy findings is given in Table 25 and Table 26.
All surviving animais treated with 1000 mg/kg/day showed thickening and sloughing of the nonglandular gastric epithelium. One male also showed a dark cranial lobe of the right lung. The female decedent showed similar stomach changes together with dark and enlarged lungs. The remaining findings in this animal were consistent with normal post mortem changes resulting from the autolytic and/or congestive processes which begin immediateiy after death.
No treatment-related macroscopic abnormalities were detected at 150 or 15 mg/kg/day.
The incidental findings recorded for a male treated with 150 mg/kg/day, identified as hydronephrosis and dark lungs, whilst showing no dose-related response, were consistent with normally expected low incidence findings in laboratory maintained rats and were of no toxicological significance.

Histopathology
There was one unscheduled death during the course of the study. A female treated with 1000 mg/kg/day died before scheduled termination. The observation of histopathologicai tracheal lesions in this animal indicates that respiratory tract changes may be implicated in morbidity.
The following treatment-related changes were detected:
Stomach: Epithelial acanthosis and hyperkeratosis were observed in the forestomach of rats of either sex receiving 1000 mg/kg/day and in female rats dosed with 150 mg/kg/day.
Trachea: Various epithelial changes characterised by atrophy/flattening, loss of cilia, hypertrophy/hyperplasia, subepitheliai inflammatory celi infiltrates, erosion, and inflammatory exudate, were observed in relation to treatment for rats of either sex dosed at 1000 mg/kg/day. The appearance of these changes is suggestive of inadvertent instillation of small amounts of an irritant test material into the respiratory tract. Occasional instances of epithelial hypertrophy/hyperplasia were also evident in other treatment groups but the significance of these is less convincing.
Lungs: Hypertrophy/hyperplasja of the bronchiolar epithelium was observed for one male and for one female rat dosed at 1000 mg/kg/day. In view of the changes elsewhere in the respiratory tract, the possibility that this unusual change is a consequence of administration of the test material cannot be excluded. Animais in the remaining treatment groups were unaffected.
Ail remaining morphologicai changes were those commonly observed in laboratory maintained rats of the age and strain empoyed, and there were no differences in incidence or severity between control and treatment groups that were considered to be oftoxicological significance.l

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
food consumption and compound intake
clinical biochemistry
histopathology: non-neoplastic
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects seen at 150 mg/kg were not indicative of serious damage to health: Salivation (rather immediate), Posture in female, histo: forestomach => "adaptation" phenomena linked to physical/organoleptic characteristics of the product during its ingestion.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material, Lipacide UG, to rats for a period of up to twenty-eight consecutive days at dose levels of 15, 150 or 1000 mg/kg/day resulted in treatment-related microscopic changes at dose levels of 1000 and 150 mg/kg/day. No such changes were detected in animals treated with 15 mg/kg/day and the “No Observed Effect Level” (NOEL) was considered to be 15 mg/kg/day.
The effects seen at a dose level of 150 mg/kg/day were not indicative of serious damage to health as defined by the European labelling guide of Commission Directive 93/21/EEC. A NOAEL value of 150 mg/kg bw/d was considered.
Executive summary:

Introduction.


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 July 1995).


 


Methods. The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis ou BP).


 


Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all surviving animals at the end of the study.


All animals were subjected to gross necropsy examination and histopathoiogical evaluation of selected tissues was performed.


 


Results.


Mortality.


One female treated with 1000 mg/kg/day was found dead on Day 24 of the study. There were no other deaths.


Clinical Observations.


Increased salivation was detected immediately before dosing and for up to one hour after dosing in animals of either sex treated with 1000 mg/kg/day from Day 3 onwards. Hunched posture and noisy respiration were evident in this dose group from Day 7 onwards, increasing in incidence as the study progressed. Incidents of pilo-erection, tiptoe gait, wet and stained fur and stained mouth were also observed during the study and by the final day of treatment four animals showed gasping and/or laboured respiration. Animals of either sex treated with 150 mg/kg/day showed increased salivation for up to ten minutes after dosing and hunched posture was observed in femaies from Day 22. No abnormaiities were detected in animais treated with 15 mg/kg/day.


Behavioural Assessment. Open-field assessment confirmed the ciinicai signs of hunched posture and respiratory pattem changes detected in 1000 mg/kg/day animais, with tiptoe gait detected in one male during Week 3. Hunched posture was observed in females treated with 150 mg/kg/day during Week 4. No treatment-related effects were detected at 15 mg/kg/day.


Functional Performance Tests.


No treatment-related changes were detected.


Sensory Reactivity Assessments.


No treatment-related effects were detected.


Bodyweight.


Reduced bodyweight gain was detected for animals of either sex treated with 1000 mg/kg/day during Weeks 3 and 4 compared to controls. During the final week two males and one female showed a bodyweight loss. No abnormal bodyweight changes were detected for 150 or 15 mg/kg/day animals.


Food Consumption.


Reduced dietary intake and food efficiency were detected for 1000 mg/kg/day animals during Weeks 3 and 4 compared to controls. No such effects were detected for 150 or 15 mg/kg/day animals.


Water Consumption.


No intergroup differences were detected.


Haematology.


No treatment-related effects were detected.


Blood Chemistry.


Reductions in total plasma protein, albumin and cholesterol were detected for males treated with 1000 mg/kg/day compared to controls. No such effects were detected for 1000 mg/kg/day females or for animals of either sex treated with 150 or 15 mg/kg/day.


Organ Weights.


No treatment-related effects were detected.


 


Necropsy.


All surviving animals treated with 1000 mg/kg/day showed thickening and sloughing of the non-glandular gastric epithelium. One male also showed a dark cranial lobe of the right lung. The female decedent showed gastric and pulmonary changes together with normal post mortem changes., No treatment-related macroscopic abnormalities were detected at 150 or 15 mg/kg/day.


 


Histopathology.


The following treatment-related changes were detected:


Stomach: Epithelial acanthosis and hyperkeratosis were observed in the forestomach of rats of either sex receiving 1000 mg/kg/day and in female rats dosed at 150 mg/kg/day.


Trachea: Various epithelial changes characterised by atrophy/flattening, loss of cilia, hypertrophy/hyperplasia, subepithelial inflanunatory cell infiltrates, erosion, and inflammatory exudate, were observed in relation to treatment for rats of either sex dosed at 1000 mg/kg/day. The appearance of these changes is consistent with small amounts of an irritant test material entering the respiratory tract during dosing. Occasional instances of epithelial hypertrophy/hyperplasia were also evident in other treatment groups but the significance of these is less convincing.


Lungs: Hypertrophy/hyperplasia of the bronchiolar epithelium was observed for one male and for one female rat dosed at 1000 mg/kg/day. In view of changes elsewhere in the respiratory tract the possibility that this unusual change is a consequence of administration of the test material cannot be excluded. Animals in the remaining treatment groups were unaffected.


 


Conclusion.


Oral administration of the test material, Lipacide UG, to rats for a period of up to twenty-eight consecutive days at dose levels of 15, 150 or 1000 mg/kg/day resulted in treatment related microscopic changes at dose levels of 1000 and 150 mg/kg/day.


No such changes were detected in animals treated with 15 mg/kg/day and the “No Observed Effect Level” (NOEL) was considered to be 15 mg/kg/day.


The effects seen at a dose level of 150 mg/kg/day were not indicative of serious damage to health as defined by the European labelling guide of Commission Directive 93/21/EEC.