Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 3 March 1995 and 25 May 1995.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health and Welfare (M.H.W.) guidelines 1986 for a 28 day repeat dose oral toxicity study.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of signature: 16/08/95 Date of inspection: 16/03/94
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification : PX-200Chemical name : Same as submission substanceBatch number : P-3Date received : 24 February 1995Description : White powderContainer : White opaque plastic jarStorage conditions : Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River (U.K.) Limited, Manston, Kent, UK.- Age at study initiation: 5-6 weeks.- Weight at study initiation: Males: 146-189 gFemales: 131-157 g- Fasting period before study: Overnight.- Housing:Animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.- Diet: Ad libitum - Rat & Mouse SQC Expanded Diet No. 1- Water: Ad libitum - mains water- Acclimation period:Not stated.ENVIRONMENTAL CONDITIONS- Temperature (°C): 21 ± 2°C- Humidity (%): 55 ± 15%- Air changes (per hr): at least 15 air changes per hour.- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light (controlled, low-intensity flourescent lighting)IN-LIFE DATES: From: Day 1 To: Day 28

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:For the purpose of this study the test material was prepared at the appropriate concentrations as a suspension in Arachis oil B.P. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Results are given in Appendix XI (attached) and show the formulations to be stable for at least ten days. Formulations were therefore prepared weekly and stored at 4 deg C in the dark.DIET PREPARATIONNot applicable, gavage study.VEHICLE- Justification for use and choice of vehicle (if other than water): Not stated.- Concentration in vehicle: 0, 12.5, 62.5, 250 mg/ml- Amount of vehicle (if gavage): 4 ml/kg- Lot/batch no. (if required): P-3- Purity:Not stated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test material formulation and were analysed for concentration of PX-200 at Safepharm Analytical Laboratory. The method used for analysis of formulation and the results obtained are given in Appendix XI. The results indicate that the prepared formulations were within ± 9% of the nominal concentration.
Duration of treatment / exposure:
28 days.
Frequency of treatment:
The test material was administered daily, for up to 28 consecutive days, by oral gavage using a stainless steel cannula attached to a disposable plastic syringe.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:0Basis:actual ingested
Remarks:
Doses / Concentrations:12.5 mg/lBasis:actual ingested
Remarks:
Doses / Concentrations:62.5Basis:actual ingested
Remarks:
Doses / Concentrations:250Basis:actual ingested
No. of animals per sex per dose:
5 males and 5 females per dose level.Concentrations 0 and 250 mg/ml were repeated as satellite groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not stated.- Rationale for animal assignment (if not random): Random.- Rationale for selecting satellite groups: Not stated.- Post-exposure recovery period in satellite groups: 14 days.- Section schedule rationale (if not random):Not stated.
Positive control:
Not conducted.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: NoDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: 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. Animls were observed immediately before dosing and one hour after dosing at weekends and on public holidays. During the treatment-free period, satellite group animals were observed twice daily, morning and afternoon (once daily at weekends and on public holidays). All observations were recorded.BODY WEIGHT: Yes - Time schedule for examinations:Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28 and, in the case of satellite group animals on Days 35 and 42. Bodyweights were also recorded at necropsy.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not feeding study.Food consumption was recorded for each cage group at weekly intervals throughout the study.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 dataWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not drinking water study.Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overty changes.OPHTHALMOSCOPIC EXAMINATION: No HAEMATOLOGY: Yes- Time schedule for collection of blood:Haematological and blood chemical investigations were performed on all surviving animals from groups 1 to 4 at the end of the treatment period (Day 28) and on all satellite group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Two repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.- Anaesthetic used for blood collection: No data- Animals fasted: No- How many animals:All surviving animals.- The following parameters were examined:Haematocrit (Hct)Haemoglobin (Hb)Erythrocyte count (RBC)Total leucocyte count (WBC)Differential leucocyte countPlatelet count (PLT)Erythrocyte indicies - Mean corpuscular haemoglobin (MCH) - Mean corpuscular volume (MCV) - Mean corpuscular haemoglobin concentration (MCHC)Clotting time (CT)CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood:Haematological and blood chemical investigations were performed on all surviving animals from groups 1 to 4 at the end of the treatment period (Day 28) and on all satellite group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Two repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.- Animals fasted: No- How many animals:All surviving animals.- The following parameters were examined:The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:Blood ureaTotal proteinAlbuminAlbumin/Globulin ratio (by calculation)SodiumPotassiumChlorideCalciumInorganic phosphorusAlkaline phosphatase (AP)Alanine aminotransferase (ALAT)Aspartate aminotransferase (ASAT)GlucoseGamma glutamyl transpeptidase (yGT)TriglyceridesTotal cholesteralTotal bilirubinCreatinineCholinesterase (CHE)URINALYSIS: Yes - Time schedule for collection of urine:Urine analysis was performed on all surviving animals from group[s 1 to 4 during week 4. Urine samples were collected over a period of approximately 16 hours, by housing the rates in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.- Metabolism cages used for collection of urine: Yes- Animals fasted: Yes- The following parameters were examined:The following parameters were measured on freshly collected urine.VolumeSpecific gravitypHProteinGlucoseKetonesBilirubinUrobilinogenReducing substancesBloodNEUROBEHAVIOURAL EXAMINATION: NoOTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes Performed on the animal killed in extremis dring the study, and on all animals killed at the end of the dosing period or, in the case of satellite group animals, at the end of the treatment-free 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 subject to a full external and internal examination, and any macroscopic abnormalities were recorded.-Organ weights:The following organs, removed from the animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:AdrenalsBrainGonadsHeartKidneysLiverPituitarySpleenNormal ranges for these organ weights are given in Appendix XVI.HISTOPATHOLOGY: Yes Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:AdrenalsAorta (thoracic)Bone and Bone marrow (femur including stifle joint)Bone and Bone marrow (sternum)BrainCaecumColonDuodenumEyesGross lesionsHeartIleumJejunumKidneysLiverLungsLymph nodes (cervical and mesenteric)Muscle (Skeletal)OesophagusOvariesPancreasPituitaryProstateRectumSalivary glandsSciatic nerveSeminal vesiclesSkin (hind limb)SpleenStomachTestesThymusThyroid/parathyroidTracheaUrinary bladderUterus
Other examinations:
None stated.
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 analyssis of variance incorporating 'F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal Wallis non-parametric analysis of variance and Mann Whitnet U-test.Probability values were calculated as:p<0.001***p<0.01**p<0.05*p≥0.05 (not significant)

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 examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITYA summary incidence of daily clinical observations is given in Tables 1 to 20. Individual observations are given in Appendix I.Animals treated with the test material showed no clinically observable signs of toxicity during the study.One intermediate dose males showed an open wound in the dorso-cervical region followed by scab formation over the wound from Day 11 until terminal kill. Such physical injuries occasionally arise when rats are housed in groups and these findings were considered not to be toxicologically important. One satellite control female showed a small scab on its right ear from Day 21 onwards and a scab on its left ear during the final four days of the recovery period. These findings were clearly of no toxicological relevance.BODY WEIGHT AND WEIGHT GAINGroup mean weekly bodyweights and standard deviations are given in Tables 21 and 22 and are presented graphically in Figures I and II. Group mean weekly bodyweight gains and standard deviations are given in Tables 23 and 24 (statistically significant differences are indicated). Individual data are given in Appendix II.There was no adverse effect on bodyweight development which could be attributed to the test material toxicity.Low and intermediate dose females showed a statistically significant reduction in bodyweight gain compared with controls during weeks 1 and 2 respectively. Bodyweight development was unaffected by treatment at the high dose level and, in the absence of any dose-response relationship, these findings were considered not to be toxicologically important. Satellite high dose males showed a statistically significant increase in bodyweight gain compared with satellite controls during week 3 but such isolated increases in bodyweight gain are considered to be of no toxicological significance.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)Not feeding studyGroup mean weekly food consumptions are given in Tables 25 and 26 and are presented graphically in Figures III and IV. Weekly food efficiencies are given in Tables 27 and 28.Animals treated with the test material showed a similar dietary intake and weekly food efficiency to controls during the study.FOOD EFFICIENCYNot examined.WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)Not drinking water study.Daily visual inspection of water bottles revealed no difference between the water intake of test animals and controls.OPHTHALMOSCOPIC EXAMINATIONNot examined.HAEMATOLOGYGroup mean values and standard deviations for test and control group animals are given in Tables 29 and 30 (statistically significant differences are indicated). Individual values are presented in Appedix III.There were no toxicologically significant changes in haematological parameters measured.High dose females showed a slightly lower erythrocyte count (RBC) than controls. None of the individual values were abnormally low (normal range: 6.7-8.2E+12/L) and, in the absence of any further evidence of an underlying condition that might affect numbers of ciculating erythrocytes, the intergroup difference was considered not to be indicative of test material toxicity. Satellite high dose females showed a slight but statistically significant increase in haemoglobin (HB), erythrocyte count, and haemocrit (Hct) compared with controls whilst males from the same dose groups showed a statistically significant reduction inerythrocyte count and an increased clotting time (CT) in comparison with controls. Again, none of the individual values were outside the normally expected range for rats of the strain and age used (normal range: female Hb 13.2-15.9g/d1, male RBC 6.8-8.4E+12/L, female Hct 37.3-45.6%, male CT 23-32 secs) and, in the absence of a similar adverse ewfect amoungst high dose animals at the end of the dosing period, these findings were considered also not to be toxicologically important.The remaining statistically significant intergroup differences involved a reduction in male haemoglobin at the indermediate dose level. In the absence of a dose-response relationship, this finding was considered to be of no toxicological importance.CLINICAL CHEMISTRYGroup mean values and standard deviations for test and control group animals are given in Tables 31 and 32 (statistically significant differences are indicated). Individual values are presented in Appendix IV.There were no toxicologically significant changes in the blood chemical parameters measured.High dose males showed a slight but statistically significant increase in triglyceride concentration comared with controls. None of the individual values were abnormally high for rats of the strain and age used (normal range: 36-171mg/d1) and the intergroup difference was considered to have arisen fortuitously, probably because of a lower than expected control group mean triglyceride concentration. High and low dose males showed a higher plasma sodium concentration than controls but there was no concomitant effect on plasma chloride and, in the absence of a similar effect at the intermediate dose level, a dose-response relationship could not be demonstrated. This finding was, therefore, considered not to be toxicologically significant.The reamining statistically significant intergroup differences involved a reduced male plasma calcium concentration and an increased female alkaline phosphatase. These findings were confined to satellite high dose animals and, in the absence of an adverse effect on these parameters amoungst high dose animals at the end of the dosing period, the intergroup differences were considered not to be toxicologically important.URINALYSISA summary incidence of urinalytical findings is given in Tables 33 and 34. individual and appropriate group mean values and standard deviations are given in Appendix V.There were no toxicologically significant changes in the urinalytical parameters measured nor were there any statistically significant intergroup defferences detected.NEUROBEHAVIOURNot examined.ORGAN WEIGHTSGroup mean absolute and relative organ weights and standard deviations for test control group animals are presented in Tables 37 and 40 (statistically significant differences are indicated). Individual data are given in Appendix VII.There were no toxicologically significant organ weight changes.High and intermediate dose females showed a slight but statistically significant reduction in absolute kidney weight compared with controls. A dose-response relationship could not be demonstrated however because intermediate dose animals showed a slightly lower group mean than high dose animals and, in the absence of any other evidence of an adverse effect on the kidney, this finding was considered not to be toxicologically significant. High dopse females also showed a slight but statistically significant reduction in absolute pituitary weight compared with controls. None of the individual values were outside the normally expected range for rats of the strain and age used (normal range: female absolute pituaitary weight 0.0057 -0.0161 g) and, as there was no statistically significant reduction in pituitary weight when expressed relative to bodyweight, this finding was considered also not to be toxicologically significant.The remaining statistically significant intergroup differences involved an increased relative brain weight, a reduced relative kidney weight and an increased relative pituitary weight. These findings were confined to low and/or intermediate dose females and, in the absence of a dose-response relationship, were considered not to be toxicologically important.GROSS PATHOLOGYA summary incidence of necropsy findings is given in Tables 35 and 36. Individual post mortem examination findings are given in Appendix VI.Two high dose males showed patchy pallor of the liver at terminal kill. One of these animals also showed accentuated lobular pattern of the liver whilst the other showed a clear, colourless fluid in the thoracic cavity. No abnormalities were detected amoungst satellite high dose males following an additional fourteen days without treatment.High dose females and animals of either sex from the remaining dose groups showed no toxicologically siginificant macroscopic lesions at terminal kill.The remaining incidental macroscopic abnormalities detected at necropsy were confined to satellite control males and, as such, were clearly of no toxicological importance.HISTOPATHOLOGY: A summary incidence of histopathological findings is given in Tables 41 and 42.All findings were entered into the ROELEE84 pathology computerisation system for tabulation and report production. Details of the grading ystem used, together with all individual animal histopathological findings, are given in Appendix VIII.No tereatment-related changes were observed.All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.HISTORICAL CONTROL DATA (if applicable)Not applicable.OTHER FINDINGSNone stated.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
250 other: mg/kg/day
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
1 000 other: mg/kg/day
Sex:
female
Basis for effect level:
other: Females dosed at 1000 mg/kg/day and males dosed at 250 mg/kg/day showed no toxicologically significant changes in the parameters measured.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Discussion:

The oral administration of PX-200 by gavage for a period of up to 28 consecutive days resulted in apparently treatment related macroscopic findings at a dose level of 100 mg/kg/day amongst two males.

Both animals showed patchy pallor of the liver at terminal kill and once of these also showed an acentuated lobular pattern of the liver. The aetiology of these hepatic changes and the toxicological importance is uncertain because there were no associated morphological hepatic lesions and laboratory investigations showed no evidence of liver dysfunction. The remaining treatment-related finding of a clear, colourless fluid in the thoracic cavity has no histopathological evidence of an adverse effect on the myocardium.

No toxicologically significant findings were evident amongst the remaining animals dosed at 1000 mg/kg./day or amongst animals of either sex dosed at 250 or 50 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material, PX-200, to rats for a period of up to 28 consectutive days , by gavage, at dose levels of 50, 250 and 1000 mg/kg/day resulted in apparently treatment-related macroscopic changes amongst two males at a dose level of 1000 mg/kg./day. Females dosed at 1000 mg/kg/day and males dosed at 250 mg/kg/day showed no toxicologically significant changes in the parameters measured and, as such the "No Observed Effect Level" (NOEL) was considered to be 250 mg/kg/day for males and 1000 mg/kg/day for females.
Executive summary:

Method

The test material was administered by gavage to three groups, each of five males and five female Sprague-Dawley CD strain rats, for 28 consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil B.P.). Two satellite groups, each of five males and five females were treated with the high dose (1000 mg/kg/day) or the vehicle alone throughout the 28 day study period and then maintained without threatment for a further fourteen days.

Clinical signs, bodyweight, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all surviving main group animals at the end of the dosing period and for all satellite group animals at the end of the treatment-free period. Urinalysis was evaluated for all surviving main group animals during the final week of dosing.

All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

Results

The results are summarised as follows:

Mortality Data:

There were no deaths which could be attributed to test material toxicity.

One high dose female was killed in extremis on Day 5 due to a damaged left forepaw.

Clinical Observations

Animals treated with the test material showed no clinically observable signs of toxicity during the study.

Bodyweight

There was no adverse effect on bodyweight development which could be attributed to the test material toxicity.

Food consumption

Dietary intake and food efficiency were unaffected by treatment with the test material.

Water consumption

Water consumption was unaffected by treatment with the test material.

Haematology

There were no toxicologically significant changes in the haematological parameters measured.

Blood chemistry

There were no toxicologically significant changes in the blood chemical parameters measured.

Urinalysis

There were no toxicologically significant changes in the urinalytical parameters measured.

Necropsy

Two high dose males showed patchy pallor of the liver at terminal kill. One of these animals also showed accentuated lobular pattern of the liver whilst the other showed clear, colourless fluid in the thoracic cavity. No abnormalities were detected amongst satellite high dose males following an additional fourteen days without treatment.

High dose females and animals of either sex from the remaining dose groups showed no toxicologically significant macroscopic lesions at terminal kill.

Organ weights

There were no toxicologically significant organ weight changes.

Histopathology

No treatment-related effects were detected.

Conclusion

Oral administration of the test material, PX-200, to rats for a period of up to 28 consecutive days , by gavage, at dose levels of 50, 250 and 1000 mg/kg/day resulted in apparently treatment-related macroscopic changes amoungst two males at a dose level of 1000 mg/kg/day. Females dosed at 1000 mg/kg/day and males dosed at 250 mg/kg/day showed no toxicologically significant changes in the parameters measured and, as such the "No Observed Effect Level" (NOEL) was considered to be 250 mg/kg/day for males and 1000 mg/kg/day for females.