Question

Final report of GLP animal study

Study Groups

The groups in the study are as follows: Group 1 (Vehicle), Group 2 (Low Dose), Group 3 (Medium Dose), Group 4 (High Dose) Group 5 (High Dose with a 14 day Recovery Period) 10 male and 10 females per group Group 6 (Toxicokinetic Group - Vehicle (5 males & 5 females) & High Dose (5 males & 5 females).

Outcomes:

1) The test item was stated to be >98% pure by the Sponsor; however no Certificate of Analysis was received.

2) Following issuing of the study plan the Sponsor asked that the higher dose of the test item be administered at 600 mg/kg/day rather than 700mg/kg/day.

3) Due to an experimental error during the study, animals from Group 1 (vehicle) were administered vehicle twice rather than once on the 14th day of dosing.

4) During acclimatisation period some females and males were housed together for a single day. Fortunately assessment of the females during the course of the study indicated that they were not pregnant and therefore this error did not influence the study outcomes.

5) Three animals that received a low dose of the test item were without water for the 24 hours before necropsy. The Study Plan stated that they were to be fasted but have access to water.

6) The humidity of the room the animals were housed in reached 90% on day 14 of dosing. The Study Plan stated limits of 30-70%.

Biochemical Analysis

Group 1: Values for all parameter were within historical and literature range

Group 2: Values for all parameter were within historical and literature range

Group 3: Values for most parameters were within historical and literature range, with the exception of slightly elevated Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) and Sorbitol dehydrogenase (SDH)

Group 4: Values for most parameters within historical and literature range, with the exception a significantly higher level of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) and Sorbitol dehydrogenase (SDH). ALT, AST and SDH were very high (ALT>AST) in the animals that were euthanised.

Group 5: For those animals that completed the study the values for all parameters, after a 14-day recovery period, were within historical and literature range.

Haematology

White blood differentials were normal in all animals.

The Haematocrit was elevated in 3 animals that received the low dose otherwise all other animals had Haematocrit levels within the normal range.

Histology

Light microscopy of tissue sections showed an increased recruitment of inflammatory cells in the liver. There were no changes in the spleen, kidney or any other organ examined.

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 animals.

The high dose group showed moderate signs of toxicity manifesting as an altered gait and lack of grooming in 5 of the 10 animals. Three animals (2 male, 1 female) from group 4 and 4 animals from group 5 (2 male and 2 female) were found to exhibit severe signs of toxicity and were euthanised between the 19th and 24th day of dosing. These animals also exhibited a decrease in body weight of 5% per day for the 3 days prior. Blood was collected from these animals and all clinical biochemistry and haematology performed.

Toxicokinetics/Dose formulation

Toxicokinetic analysis indicated that mean Cmax, AUClast& T1/2 for the test item were comparable between males & females on Day 1.

Both Cmax & AUClast increased on Day 28, with mean values consistently higher for males compared to the corresponding females. T1/2 remained similar to Day 1 values for both males and females.

Analysis of the dose formulation indicated that the doses administered were as stated in the study plan.

Question

Formally record one amendment & one deviation that occurred during the course of the study.

(Using OECD GLP # 1 & OECD Guideline 407 to assist you)

Answer 1

According to the information, toxicity analysis of the molecule of interest is being done. However, the study has some major issues and deviations which are required to be recorded and amended before concluding any scientific merit.

1. The certificate of analysis is mandatory for conducting the studies in order to ensure that the molecule of interest is devoid of any toxic components.
2. Fasting of animals requires maximum 24 hours of non-eating/drinking period. Since the animals were out of water for nearly 72 hours, it might have affected the mode of action of the molecule as well as disturbed homeostasis.
3. The lowering of dose of the molecule of interest in last group for 14 day toxicity and recovery period does not comply with the standard procedure. Further, the observation that these animals demonstrated disturbed hepatic homeostasis states possible hepato-toxicity.
4. The toxicokinetics stating different plasma kinetics for male and female organisms is unacceptable and hence requires reproductive toxicity analysis to be performed in female rats as well as male rats.