Introduction
This is a biot management lab report of rat GLP which was requested by Dr Craig Smith of new technologies limited (Smith, 2000). The study was conducted in accordance to OECD guideline 407. The pharmacokinetic profile of the test item, LAF-001 had been performed and the highest dose to be assessed was 700mg/kg. The vehicle for the test item was water and the test item was in powdered form, 98% pure and was stored below -20°C and had an expiry date of Aug 2011.
A toxic analysis of plasma samples was done on the last day of dosing. The time periods for the intravenous blood collection were 10, 30, 60 min, 4, 8, 24h post-dose. The sponsor had had the tests done in a lab in US and they asked that the samples be repeated that lab. The groups for the study were: group1 (vehicle), group2 (low dose), group3 (medium dose), group4 (high dose), group5 (high dose), with a 14 day recovery period, 10 males and 10 females per group and finally group6- toxiciokinetic group only (vehicle and high dose administration).
The study in this case was able to give information on any possible health hazards that may arise as a result of repeated exposure over a comparatively repeated time period. The method comprised basic repeated dose toxicity study. The outcomes for histology indicated that light microscopy of tissue sections increased recruitment of inflammatory cells in the liver. There were no changes in the spleen, kidney, or any other organ examined. The change in daily body weight was decreased in animals on the higher dose compared to animals administered vehicle or lower doses of the test item. Toxic kinetic analysis indicated that the profile (the Cmax and t ½) for the test item did not differ. Analysis of the dose formulation indicated that the doses administered were as stated in the study plan.
Aim
The aim of the study was to test LAF -001, the oral toxicity of repeated dose in rodents to determine the effect of levels of toxicity to changes in their activities, body functioning, and body weight, both males and females.
Methodology
Sampling
The sample was ten rats, five male and five female which were not older than 9weeks. Sampling was done on the last day of the test period. The time periods for the intravenous blood collection were 10, 30, 60 min for pre dose and, 4, 8, 24hours for post-dose. The females were nulliparous and non- pregnant. All the rats were of the same species.
Housing and feeding
The environmental conditions on which they were exposed to were regulated. Humidity was between 30% and 70% but during cleaning it was kept at 50% to 60% while temperature was between 19°C and 25° C. An artificial source of light was used which was made available for 12 hours and 12 hours of darkness.
Feeding of the rats followed a special diet. Every 7days, water was changed and new water supplied. Temperature was between 2° C and 8°C to make sure that the dose does not interfere with normal nutrition and water. The dose was administered to the rats every day for 28 days. The dosage was 600 mg/kg, given at scheduled time. The dose vehicle was water. Animals were kept in cages in small groups of the same sex. There were two cages and each cage housed five females and the other five males.
Animal preparation
Care was taken to select only young rats with good health, with the assignment of the rats to their respective control groups as well as treatment groups being random. Their cages were specially arranged in a way to reduce cage placement effects. The animals underwent a unique identification and later confined in their different cages for five days prior to the commencement of this study to enable the animals to acclimatize to the conditions in the laboratory.
Dose Preparation
The compound for the test was given by gavages through drinking water, where the test substances dissolved in the water.
Procedure
Test item
The test item was ten rats, five male five females, from the same species. The rats were young healthy adults that were not older than 9weeks. The females were not pregnant to ensure that they did not affect the results. On weight, it was ensured that it was not more or less than 20% of the mean (male and female).since there were planned interim kills, the number of rats was increased by ten more, five males and five female.
Dosage
Dose levels were selected taking into account kinetic toxicity. The highest dose level was chosen to induce toxic effects. Thereafter, a reducing sequence of dose levels was selected in order to show any dose related response and.two, to four fold intervals were used for descending dose levels. A fourth test group was used between dosages.
Ad ministration doses
The rats were dosed with the test substance everyday for a period of 28 days. The volume used was 1ml/ 100g body weight. The times to give this dose was kept constant each day; adjustments were however made if and only if the dose level was to be maintained.
Test Principles
For 28 days, the rats orally received one dose level. They were then combined with those that died earlier in the study and necropsied.
Observation
The observation period was 28 days. Clinical observations were made once daily, at the same time. In addition, the animals were closely watched for any signs of mortality as well as morbidity at least once a day. Before their first exposure, a comprehensive clinical observation was made in all the rats. This was done at the same time outside the home cage. Changes in gait, posture, steriotyppies, or bizarre behavior was also recorded.
Food/water consumption and body weight
Weighing of the rats, as well as the measurement of water and food intake, was done once every week.
Hematology
The sample was kept under appropriate conditions.
Clinical biochemistry
In bio-chemistry, the measure of toxic effect included sodium, potassium, glucose, total cholesterol, creatinine, urea, total albumin and protein, ALT and AST enzyme, GGT, and bile acids (Crofton et al., 1991). Organs such as kidney, liver, and spleen necropsied very fast and the tissues were preserved by appropriate fixation method. Full histopathology examination was extended to the entire rats test. This was done in order to determine their major effects in tissues and more especially on kidney and liver. This was performed on all the blood samples obtained from all the rats, just before killing them.
Prior to blood sampling, the rats underwent an overnight fasting.
Gross Necropsy
The adrenals, liver, kidneys, testes, thymus, epididymides, spleen, brain, and heart of all rats under study were trimmed of any adherent tissue appropriately and weighed immediately after dissection to avoid dying.
Histopathology
All gross lesions were examined, and histopathology performed on tissues and organs that showed effect in the treated groups.
Discussion
General Observations
The vehicle and low dose groups exhibited no signs of toxicity. The mid level group showed mild to moderate signs of toxicity manifesting as an altered gait and lack of grooming in 3 of the 10 rats of each sex. The high dose group showed moderate signs of toxicity manifesting as an altered gait and lack of grooming in 5 of the 10 rats. Three rats (2 male, 1 female) from group 2 and 4 rats from group 1 (2 male, 2 female) were found to exhibit severe signs of toxicity and were euthanized between 19th and 24th day of dosing. These rats also experienced a loss in body weight of 5% per day for three days. Blood was collected from these rats and all clinical hematology and biochemistry performed. The change in daily body weight decreased in animals on the higher dose compared to rat administered vehicle or lower doses of the test item.
Toxic kinetics/ Dose Formulation
Toxic kinetic analysis indicated that the profile (Cmax and T1/2) for the test item did not differ. Dose formulation analysis indicated that the doses administered were as stated in the study.
Biochemical analysis
Group 1 and 2 values for all parameters were within historical and literature range. In group 3, most of the parameters were within historical and literature range, except slightly elevated alanine aminotransferase (ALT, Aspartate aminotransferase (ASP) and Sorbitol dehydrogenase (SDH). In group 4, values for most parameters were within historical and literature range except significantly high levels of ALT and AST, ALT, AST, and SDH were very high (ALT>AST) in the rats that were euthanized. For the rats that completed the study, parameter values were within historical and literature range.
This type occurs between two or even more persons with a specific person communicating with the others. The message here emanates from a specific individual. It can be done by a person to an audience, or face to face, post, couriers, mobile messages, telephone, or through emails.
It can be classified further as communications from social contacts, expert and advocates. When the company’s sales persons communicate to the customers, this is referred to as communication from the advocates of that product. Expert communication occurs when an independent expert communicates to the prospective buyers about the advantages of the product while social channel communication occurs when a neighbor talks positively about a given brand.
Haematology
White blood differentials were normal in all animals. The haematocrit was elevated in 3 rats that received the low dose but all the other rats had haematocrit within the normal range.
Histology
Light microscopy of tissue sections showed an increase in recruitment of inflammatory cells in the liver. There were no changes in the kidney, spleen, or any other organ examined.
Conclusion
The test item was >98% pure as stated by the sponsor but no certificate of analysis was received. The sponsor also asked that the higher dose of the test item be administered at 600mg/ kg/ day instead of 700mg/ kg/day. Due to experimental errors during the study, rats from group 1(vehicle) were administered vehicle twice instead of once on the 14th day of dosing. In period of acclimatization, some males and females were housed together for a day and fortunately, assessment of the females during the study indicated that they were not pregnant and hence did not affect the outcomes of the study. Three rats that received low dose of the test item were not given any water for 24 hours before necropsy. The humidity of the room the rats were housed reached 90% on the 14th day of dosing.
This is where there is no direct link between the sender and the receiver of the messages. It is also called non-personal form of communication and it includes the use of events, the atmosphere and media. In this case, atmosphere refers to what a firm creates in their different offices. The office exteriors and interiors have a great meaning to potential customers.
Media channels here are the broadcast media (television, radio), the print media (souvenirs, magazines, proceedings of conferences, newspapers), electronic media and display media. Events can be defined as occurrences planned to communicate specific messages to target audiences. For example, a company may set opening ceremonies of different kinds, sponsorships of different events and news conferences.
Recommendation
Experimental errors during the study should be avoided by ensuring that everything taking place during the study and in the lab is recorded. Before undertaking any activity, all records should be reviewed to avoid doing mistakes like the double administration of the vehicle or test substance. According to Tupper and Wallace (1980), males and females should also never be housed together. Further more, cage temperatures, light and humidity should be strictly controlled at the recommended level (Gad, 1991).
References
Crofton K.M., Howard, J.L., Moser, Gill, M.W., Reiter, L.W. and Tilson, H.A., (1991). Interlaboratory Comparison of implications of neurotoxicology. Neurotoxicol, Teratol. 13, 599-609.
Gad, S.C. (1982). A Neuromuscular Screen for Use in Industrial Technology. Macquarie University, Sydney.
Smith, C. (2000), Chairman’s report of the meeting of the hoc working group of.
Tupper, D.E., Wallace, R.B. (1980) Utility of the Neurologic Examination in Rats. In Oxford Textbook of Public health. 4th edition. Edited by: Detels R, McEwen J, Beaglehole R, And Tanaka H. New York, Oxford University Press Inc:829-63.