OECD-GLP Guidelines: Toxicological Tests Report

Abstract

The study was conducted in accordance with the OECD guidelines on Good Laboratory Practice for non-clinical studies involving animals. The test substance (LAF 001), which was supplied by Dr. Smith (the sponsor) was administered orally using water as the vehicle to six groups of rats. The duration of exposure was 28 days. During and after the duration of exposure, the observational studies, biochemical analyses, hematological tests, and histological studies that were conducted on the rats show that the biological responses in the rats did not differ significantly from the historical and literature values. Except for a few isolated cases, the study conforms to the OECD guidelines on GLP for toxicological tests (OECD 1998a).

Introduction

Good Laboratory Practices (GLP) entail a set of guidelines as provided by the Organization for Economic Co-operation and Development (OECD), FDA, and the EU with the aim of ensuring that the studies conducted by research institutions and their affiliate laboratories are consistent and reliable (OECD 1998a, OECD 1998b, OECD 1998c, OECD 1998d). In most cases, GLP affects the non-clinical studies meant to evaluate the probable hazards and possible risks involved in releasing materials, chemical substances, and drugs to human beings, animals, and the environment (Crissman 2004, p. 206; Morton 2006, p. 806). The test substances used in these studies include pharmaceuticals, detergents, biocides, food additives, contaminants and many more (Webster 2005, p. 182).

Aim

Considering that the acute toxicity studies on the test item had been conducted earlier and the highest dose determined, it is possible to conduct the oral toxicity studies using repeated doses (Comstock 2008, p. 1010). Thus, the current study was aimed at determining the possible health hazards expected from repeated exposure of rats to the test item.

References

The current study plan was prepared and used in accordance with the OECD Guideline 407 for the Testing of Chemicals, which was adopted on the 27^th July 1995. This guideline provides information on the protocols and considerations used in repeated dose (for 28 days) oral toxicity studies in animals particularly rodents (OECD 1998b; OECD 1998d). The same protocols were replicated in the current study involving rats.

Test Substances and Controls

The sponsor, Dr. Craig Smith, provided the test item together with the required information. According to OECD (1998a; 1998b), the Sponsor took full responsibility in terms of the description and identification of the test item with the exclusion of confidential and trade-related information as follows:

Test Item

Name of the test item: Not specified

CAS Number: Not Provided

Batch Number: LAF 001

Physical Properties: 98% Pure Powder

Color of the test item: Not Provided

Expiry Date: August 2011

Solubility: Highly soluble in water, thus water was used as the vehicle for the test item

pH: Not Provided

Density: Not Provided

Stability: The test item is stable in water for approximately 7 days at a temperature of 2-8°C

Storage: The item should be stored at a temperature below -20°C

Safety Considerations: Not Provided

The Highest Dose: 600mg/ kg/ day.

Controls

There were no reference test items provided by the sponsor and thus the vehicle was used as the control item. However, in most cases a reference test item is necessary for comparisons (Vishwanathan 2005, p. 23; OECD 1998d, p. 8)

Study Design and Dose Levels

Test Animals

The species of the rats used in the study were not specified as required (Comstock 2008, p. 1015). However, the rats were divided into six different groups. Each group of rats was made up of 10 females and 10 males (Weil 2007, p.179). In addition, the following criterion was used in selecting the rats suitable for the study:

Health Status: All the rats were young and healthy

Sex: All females were nulliparous and non-pregnant

Age: The rats chosen were less than nine weeks old for both males and females.

Body Weight: Both males and females weighed ≥ 20% of the average weight (OECD 1998a, p. 5).

Environmental Conditions of the Test Facilities

The investigation room was kept at a temperature range of 19°C-25°C and humidity range of 30% to 70% (Weil 2007, p. 177). However, during the process of cleaning the room, the humidity was kept at a range of 50% to 60%. On the other hand, the animals were supplied with artificial lighting in which 12 hours of darkness and 12 hours of light were allowed (OECD 1998c; OECD 1998b).

Animal Caring and Feeding

The animals were supplied with specified diets and a regular supply of water. In addition, the food given to the rats was changed every seven days at a temperature of 2-8°C (OECD 1998b, p. 4).

Dose levels

The animals were dosed on the basis of their group numbers as follows:

1. Group 1: Vehicle
2. Group 2: Low dosage levels
3. Group 3: Medium dosage levels
4. Group 4: High dosage levels
5. Group 5: high dosage levels with a 14 day recovery period
6. Group 6: Vehicle and high dosage levels for toxicokinetic studies (OECD 1998a)

In addition, dosing of the test item was done on a daily basis for 28 days. The highest dose as specified by the sponsor was 600 mg/ kg of body weight per day. Moreover, dosing was done at the same time each day.

Experimental Evaluation

Observations

Observations were made for a period of 28 days during which time the rats were left without any treatment for 14 days. This is important because it enables the analyst to evaluate the possibility of the rats recuperating from the toxic effects of the test item (Webster 2005, p. 185). In addition, general clinical observations were conducted once per day to examine the climax stages of the anticipated outcomes after dosing. On the other hand, a general health observation was conducted twice per day to assess any cases of mortality and morbidity. Most importantly, a major clinical examination was conducted on all animals before the first exposure and once per week after exposure (OECD 1998a).

Measurement of Changes

Any major observable changes in the skin, fur, eyes, mucous membranes, the presence of excretions and secretions, and other autonomic activities were recorded on a daily basis. In addition, any changes in posture, gait, response, the presence of tonic movements, and bizarre manners were also recorded. On the other hand, the rate of water consumption and changes in the body weight were measured on a weekly basis (OECD 1998a; OECD 1998b).

Sampling Times

Sampling involved collection of blood samples from the test animals. The pre-dosing blood samples were collected at 10, 30, and 60 minutes before dosing. On the other hand, the post-dosing blood samples were collected at 4, 8, and 24 hours after dosing. All the samples were collected at the end of the test period (OECD 1998b).

Testing

Hematological tests were conducted on the blood samples to measure the hemoglobin concentration, total and differential leukocyte count, erythrocyte count, the blood clotting duration/potential, and the platelet count (Society of Toxicologic Pathologists 1991, p. 290). On the other hand, biochemical analyses were carried out to evaluate the toxicity levels in the kidney and the liver. The samples used for the biochemical analyses were collected after an overnight fasting period (OECD 1998d).

Moreover, histological studies on the kidney, spleen, and other organs were also carried out. On the other hand, toxicokinetic analyses were conducted on the last day of dosing on pre-dose blood samples collected at time points of 10, 30, and 60 minutes and the post-dose blood samples collected at time points of 4, 8, and 24 hours after dosing (OECD 1998a; OECD 1998b).

Results

Outcomes

Test Item: The sponsor had hinted that the test substance was less than 98% pure but failed to attach a certificate of analysis.

Highest Dose: After the sponsor was supplied with the preliminary study plan, he decided that the highest dose should be 600 mg/kg/day instead of the afore-mentioned 700 mg/kg/day.

Experimental Error: Due to an error that occurred during the study, rats from Group 1 (Vehicle) were given two doses of the vehicle instead of one on the 14^th day of dosing.

Acclimatization Period: During this period, some females and males were held together for one day. However, this did not alter the outcomes because the females did not get pregnant.

Necropsy: Before necropsy, three rats from Group 2 (Low dose level) stayed without water for a period of 24 hours.

Humidity: The humidity in the investigation room rose to 90% on the 14^th day of dosing.

Biochemical Analysis

• Group 1: The measure of all parameters considered was within the historical and literature range of values.
• Group 2: The measure of all parameters considered was within the historical and literature range of values.
• Group 3: For most of the parameters considered, their values were within the historical and literature range. However, slight elevations of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and Sorbitol dehydrogenase (SDH) were recorded.
• Group 4: The values recorded for most parameters were within the historical and literature range except for the levels of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and Sorbitol dehydrogenase, which were slightly higher. On the other hand, the values of ALT, AST, and SDH were very high in euthanized animals with their ALT being more than AST.
• Group 5: For the animals that survived the exposure to the test item in Group 5, the values for most parameters were within historical and literature range.

Hematological Studies

White Blood Cells: The white blood cell differentials were within normal levels in all animals.

Haematocrit: Three animals from Group 2 (Low dose) recorded an elevated Haematocrit while all other animals had their Haematocrit levels within the normal range.

Histological Studies

Microscopy: Observation of tissue sections under a light microscope showed an increased number of inflammatory cells in the liver. Otherwise, there were no major changes in other tissues such as the kidney, spleen, and other organs.

General Observations

Group 1 and 2: There were no signs of toxicity exhibited in the two categories.

Group 3: Rats in this category had mild to moderate signs of toxicity, altered gaits and the absence of grooming in 3 out of 10 animals observed.

Group 4: Rats in this category showed moderate signs of toxicity, altered gaits and lack of grooming in 5 out of 10 animals observed. In addition, 3 rats (2 males and 1 female) in Group 4 and four rats (2 males and 2 females) in Group 5 exhibited severe signs of toxicity and thus, they were euthanized between the 19^th and 24^th day of dosing. Moreover, clinical biochemistry and hematological studies were performed on blood samples collected from the euthanized animals.

Body Weight: Animals on higher doses exhibited lower changes in body weight compared to animals on the Vehicle dose and the lower doses of the test substance.

Toxicokinetics/ Dose Formulation

The toxicokinetic analyses indicated that the toxicokinetic profile (the Cmax and T1/2) of the test item had no significant difference. On the other hand, the dose formulation analysis indicated that the doses administered were as provided in the study plan.

Analysis and Discussions

The study was aimed at assessing the biological response of the rats to the test item (LAF 001). From the discussions above, it can be deduced that the study complies with the good laboratory practice principles as provided by the OECD and FDA. Besides, the organization of the study facilities, personnel, identification of the test systems, and the experimental design, dosage levels, collection of samples indicates compliance to the OECD-GLP guidelines (OECD 1998a; OECD 1998b). Moreover, the study was conducted in accordance with the OECD study plan for repeated dose oral toxicity testing in animals (rodents).

However, the study deviates from some of the principles of GLP involving the responsibilities of the sponsor such as provision of the certificate of analysis to support the claims made on the test item and its characterization. In addition, the study and the study facility in general demonstrated lack of a Quality Assurance programme and failure to incorporate a reference test item in the study.

Recommendations for Future Improvements in the Study

Despite that the study is guided by the OECD-GLP guidelines, several shortcomings are notable in terms of the basic principles of Good Laboratory Practices. Therefore, it hereby recommended that future studies should put the following items into practice to realize consistency and reliability in the results.

• Development of a proper Quality Assurance Programme: This entails all the activities carried out to ensure that confidence and reliability are established in the results obtained and the reports generated. These activities include use of the Standard Operating Procedures (SOP’s) in all aspects of the study, development of statistical methods for data analysis, calibration of materials and equipments before use, certification of reagents and study materials, certification of personnel, and sample tracking (OECD 1998c; Society of Toxicologic Pathologists, 1997, p. 655).
• Incorporation of the reference test item or negative control into the study: The reference item refers to a material, which exhibits the same potency as the test item. In addition, the properties and the toxicological test results of the reference test substance are known. Therefore, making comparisons between the results generated from the two materials enables analysts to come up with consistent and reliable results for the unknown test item (OECD 1998b).

Conclusions

The study examines the biological response and any anticipated risks and outcomes of the test substance on rats relative to the OECD’s guidelines on the Good Laboratory Practices. Relative to the experimental design and the test results, it can be deduced that the study complies with some of the principles of GLP. On the other hand, the test item (LAF 0001) did not produce any significant toxicity or severe biological changes in the rats except for a few isolated cases.

Reference List

Comstock, JM 2008, ‘Good Laboratory Practice and Copyright: an agrochemical perspective,’ ACS Symposium Series, vol. 369, no. 1, pp. 1010-1021.

Crissman, JW 2004, ‘Best practices guideline: toxicologic histopathology,’ Toxicol Pathol, vol. 32, no. 1, pp. 126–131.

Morton, D 2006, ‘Best practices for reporting pathology interpretations within GLP toxicology studies,’ Toxicol. Pathol., vol. 34, no. 6, pp. 806-809.

OECD 1998 a, ‘Principles of Good Laboratory Practice (as revised in 1997),’ OECD Environmental Health and Safety Publications, Vol. 1, no. 3, pp. 45-64.

OECD 1998 b, ‘OECD principles on Good Laboratory Practice,’ OECD Guidelines for the Testing of Chemicals, vol. 3, no. 1, pp. 1-14.

OECD 1998 c, ‘Quality Assurance (QA) and GLP,’ OECD Guidelines for the Testing of Chemicals, vol. 6, no. 1, pp. 6-8.

OECD 1998 d, ‘Guidance for the preparation of GLP inspection reports,’ OECD Guidelines for the Testing of Chemicals, vol. 2, no. 1, pp. 23-34.

Society of Toxicologic Pathologists 1991, ‘Peer review in toxicologic pathology: some recommendations,’ Toxicol Pathol, vol. 19, no. 3, pp. 290–292.

Society of Toxicologic Pathologists 1997, ‘Documentation of pathology peer review,’ Toxicol Pathol, vol. 25, no. 6, pp. 655.

Vishwanathan, CT 2005, FDA perspectives on current issues in GLP: in Presentation at Society for Quality Assurance Regulatory Forum, Wiley Publishers, Baltimore, M).

Webster, GK 2005, ‘JALA Tutorial: Considerations When Implementing Automated Methods into GLP Laboratories,’ Journal of the Association for Laboratory Automation, vol. 10, no. 3, pp. 182–191.

Weil, CS 2007, ‘Selection of the valid number of sampling units and a consideration of their combination in toxicological studies involving reproduction, teratogenesis or carcinogenesis,’ Food and Cosmetics Toxicology, vol. 8, no. 2, pp. 177-182.