Discussion on the Quality of Organs in the Safety Evaluation of Preclinical Drugs

The evaluation of organ quality in toxicity studies is an important part of the evaluation of drugs, compounds and medical devices. The American Society of Toxicology and Pathology (STP) has made corresponding guidelines and recommendations for organ weighing in general toxicity studies, and detailed the value of weighing data for major organs in special systems such as the endocrine system, immune system, and reproductive system. Explanation.
It is very important to adopt appropriate anatomical experimental programs according to the different test products, and it is very important to selectively increase or remove the weighing of the organ quality. Regardless of the type of research or organ to be evaluated, it is best for pathologists to comprehensively consider the type of compound in the study, the mechanism of action, and the overall data setting when evaluating the quality of the organ.
Explains the standardization of organ quality collection, the organ quality that needs to be collected in different systems, and the interpretation of organ quality data. This will provide support and support for the scientific evaluation of organ quality data in non-clinical drug safety research in my country.

The change of organ quality is a sensitive indicator of the changes in tissues and organs caused by exogenous compounds or biological agents. Therefore, the evaluation of organ quality is an important part of the toxicological evaluation of drugs, compounds, biological products, food additives and medical devices. At present, the evaluation of organ quality has been widely used in preclinical drug safety evaluation research.
In 2007, the American Society for Toxic Pathology (STP) conducted investigations on the quality of organs in the toxicity studies of multiple multinational pharmaceutical companies, animal health research institutions, and compound, food or nutraceutical product manufacturers, and the results showed that they were different There are obvious differences in the organ weighing operations of companies or institutions.
In the same year, the United States STP made corresponding guidance and recommendations on the weighing of organs for general toxicity studies in the good laboratory practice (GLP) of non-clinical drug research for 7 days to 1 year. This guiding principle discusses in detail the organ quality collection and general toxicity studies that need to be considered for the collection of organ quality, the analysis of organ quality data, and the interpretation of organ quality data.
In recent years, the requirements and data interpretation of organ quality weighing for different systems such as the reproductive system and immune system have once again caused extensive discussion among pathologists [4-5]. Although toxicologists and pathologists agree that the weighing of organ quality is an important means of determining changes in treatment factors, there are different views on which organ quality is more valuable. In toxicity studies, liver, kidney, and testis are routinely weighed, and most scholars also recognize that the quality of these organs is of great value.
Other organs that are routinely weighed include the brain, adrenal glands, ovaries, thyroid, uterus, heart, and spleen. However, the lungs, lymph nodes, and other reproductive organs are not often weighed in routine toxicity studies. They are only weighed in some special studies, such as respiratory studies, immunotoxicity studies, and reproductive studies. Of weighing.
In addition, the body mass of the organ is calculated more than conventionally, because the value is more meaningful when the body mass changes. However, the ratio of organs to brain is calculated by most North American companies, but veterinary products or European companies rarely do calculations. In short, the value of organ quality assessment of different individuals, the description of organ quality data, and the value of statistical analysis are the focus of attention of toxicologists and pathologists.
This article will discuss the standard collection of organ quality, organ quality in general toxicity studies, the value of different organ quality assessments, and the focus of organ quality data interpretation in order to improve the preclinical safety of drugs in my country. In the evaluation research, standard organ quality weighing and scientific and rigorous interpretation of organ quality data are used to provide support, and to provide protection for the safety of Chinese people’s drug use.

1.Collection of organ quality data

The order of laboratory animal anatomy is very important for obtaining meaningful organ quality [3]. During the dissection, it is necessary to avoid that the animals dissected by a certain dissecting technician come to the same dose group, because there are differences in the extraction and trimming of tissues and organs by different dissecting personnel, which will affect the test results. Therefore, the order of anatomy should be randomly or appropriately circulated to reduce trial errors. The organs must be separated gently, removing irrelevant surrounding tissues. If the organ does not need to be weighed, it should be placed in the fixative immediately after being isolated.
If the separated organs need to be weighed, they should be kept moist. You can spray saline at an appropriate time and put them in the fixative immediately after the weighing, because some small organs may be dehydrated quickly, which affects the quality of the organs. Paired organs are usually weighed together. The quality of perfused fixed organs changes due to the perfusion of fixative or other liquids, so the quality of perfused organs cannot be compared with the quality of fresh organs and immersed fixed organs. The difference in the volume of the contents will affect the quality of the organs, so the contents need to be removed. For example, the gallbladder should be incised before weighing the liver.
The final body mass weighing method of all animals in the same toxicity study should be the same. Theoretically, the final body mass is an important factor in the calculation of the organ mass ratio. It should be weighed at the time of dissection instead of on the morning of the day of the dissection. This can reduce the deviation of animal body mass caused by the difference in stress and diurnal fluctuations. . If the rodents are fed before the scheduled necropsy, or the dissection of the study will continue for one or more days, then it is very important to use the above-mentioned terminal body mass weighing method.
Dead animals are generally considered to be unnecessary for organ weighing. Because the animal’s nutritional status, bloodletting, tissue congestion and edema are quite different at this time, and there is a lack of data from the control group animals in the same period, this complicates the interpretation of the quality of the organs collected by the dead animals and cannot directly reflect the processing factors. Influence.
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2.Organ quality in general toxicity studies

2.1 Guidelines and requirements for organ weighing in general toxicity studies
There are some differences in the recommended practices for organ quality in the guidelines for management practices in different countries, and there is a lack of uniform standards. The Red Book of the Center for Food Safety and Applied Nutrition (CFSAN) of the US Food and Drug Administration (FAD), the organ quality opinion paper recommended by the US STP, and the guidelines of the Organization for International Economic Cooperation and Development (OECD), Japan’s Ministry of Health, Labour and Welfare (MHLW) guidelines on different rodent and non-rodent toxicity studies that require specific weighing of the organs are listed in Table 1. The organ quality proposed in the CFSAN Red Book includes 12 organs, namely brain, heart, liver, spleen, kidney, adrenal gland, thymus, thyroid, testis, epididymis, ovary, and uterus.
The requirements for organ quality in the MHLW guidelines are similar to those of CFSAN. The difference is that the prostate must be weighed, while the uterus, thymus, and thyroid are not required to be weighed. In addition, the MHLW guidelines recommend that the lungs, salivary glands, thymus, thyroid, uterus, and seminal vesicles are also routinely weighed organs. The routine weighing organs recommended by STP in the United States include liver, heart, kidney, brain, testis (preferably sexually mature animals), and adrenal glands. All species except mice need to weigh the thyroid and pituitary. Rodents also need to weigh the spleen and thymus.
The requirements for organ quality in the Technical Guidelines for Drug Re-dose Toxicity Tests of the Center for Drug Evaluation (CDE) of the China National Medical Products Administration are basically consistent with the guidelines of CFSAN in the United States and OECD in Europe. Although the requirements for weighing reproductive organs (such as epididymis, seminal vesicle gland, prostate, etc.) and endocrine organs (pituitary gland, thyroid) are still slightly different in different countries, all important organs need to be weighed. Therefore, according to the different compounds, combined with the corresponding guidelines, the selection of appropriate organs for weighing is essential for scientifically and accurately assessing the toxicity of drugs.
The guidelines and requirements for organ quality weighing in various countries are shown in Table 1. What needs to be explained is: X stands for weighing, Xrm in the STP recommendation letter indicates that only rats and mice need to be weighed, and Xrp is in the STP recommendation letter. In indicates that only rats and non-human primates need to be weighed, Xr indicates in the STP recommendation that only rats need to be weighed, and Xnr indicates in the OECD guidelines that “the thyroid contains parathyroid glands are weighed together”, Xa In the MHLW guidelines, it means “weighing is always required”, Xo in the MHLW guidelines means “weighing is often required”, and Xto in the MHLW guidelines means “we always weigh only the thyroid without parathyroid glands”, Xso In the MHLW guidelines, it means “weigh only the seminal vesicle glands”, and X* in the CDE guidelines indicates that only rodents need to be weighed; CFSAN selects the organs to be weighed here from its Red Book of subchronic, carcinogenic The guideline of the 1-year toxicity study. The organs to be weighed by STP are selected from the recommendations of the general repeated dosing toxicity study (duration 7 d to 1 year). The organs to be weighed by the OECD are selected here. From the guidelines for the 90-day repeated oral administration of rodent toxicity studies, MHLW selected the organs to be weighed from the guidelines for its absolute and relative body mass organ quality. The CDE selected requirements at this location. Weighing the organs comes from the technical guidelines for repeated drug administration toxicity tests.

2.2 The focus of routine organ weighing in general toxicity studies
Changes in organ quality are usually related to processing factors. The selection of appropriate organ weighing in toxicity studies requires in-depth understanding of the mechanism of action of the test substance, relevant pharmacokinetic and toxicological data, the physiological state of experimental animals, and the background data of the same or similar compound research in the previous period. STP believes that organ weighing should be routinely performed in general toxicity studies of repeated dosing. The mass value should be recorded in metric units.
Changes in the quality of different organs may indicate certain toxic reactions related to the treatment factors. Changes in liver quality may be related to hepatocyte hypertrophy, such as enzyme induction or peroxisome proliferation. For studies of less than 7 days of effective liver enzyme inducers, liver mass weighing can also be evaluated. The increase in heart mass may be the only evidence of myocardial hypertrophy, because this change is difficult to find on gross and histopathological examinations. The changes in the kidney may reflect renal toxicity, such as renal tubular hypertrophy, hyperplasia or chronic kidney disease [10]. Changes in the quality of the adrenal glands may show hypertrophy, hyperplasia or atrophy related to stress, endocrine or the effects of the test substance.
Changes in brain mass are rarely associated with neurotoxicity. Brain mass is mainly used to calculate the ratio of organs to brain. Some experts believe that the organ-to-brain ratio is very meaningful when the animal’s body mass changes too much at the end of the experiment. Or, when the difference between individual animals is too great, the organ-to-brain ratio can be calculated to standardize the organ quality. Therefore, STP recommends weighing the mass of the brain in order to calculate the mass ratio of the organs to the brain if necessary.
2.3 Weighing the organs of the endocrine system
When the thyroid and pituitary gland are properly weighed, the changes in their quality may reflect endocrine disorder, and this disorder may not be immediately manifested in histopathological examination. STP recommends that the quality of the thyroid and pituitary gland should be routinely checked in toxicity studies in animals other than mice. In principle, the quality of the mouse’s thyroid and pituitary has similar values, but the process of extraction and weighing is very easy to cause artificial damage, which makes the thyroid and pituitary microscopy difficult, so the weighing of the mouse’s thyroid and pituitary is Selective but not necessary. Fixing the thyroid and pituitary before weighing can provide more accurate weighing data and can also maintain better morphological characteristics. Therefore, it is better to collect the thyroid and pituitary by weighing after fixation for rodents. Most pathologists believe that thyroid and pituitary weighing are meaningful because the difference between animals is small and closely related to histopathological changes, which is a sensitive indicator of toxicity prediction. In particular, many researchers believe that thyroid quality is very sensitive to the reduction of thyroxine levels, so it can be an observation indicator of liver enzyme induction together with liver quality and liver morphological changes.
2.4 Weighing of reproductive organs
The quality of the testicles of sexually mature animals has a certain value. Changes in testicular quality may reflect the edema of seminiferous tubules or interstitial. Changes in the quality of the epididymis may be a more sensitive indicator of sperm reduction, and an increase in the quality of the epididymis may reflect the edema or inflammation of the epididymis. The changes in the quality of the prostate may be related to the changes in drug-induced hormone levels, that is, the drugs cause changes in androgen or estrogen signals, resulting in changes in the quality of the prostate. For repeated-dose toxicity studies in rats, the testis, epididymis, and prostate are usually sexually mature organs and should be weighed routinely. In mice, the testis should also be weighed, while the prostate and epididymis can be weighed according to different research requirements. The quality of the seminal vesicle glands provides similar information to that of the prostate. Therefore, if there are data on the quality of the prostate, weighing the seminal vesicle glands is of little significance for toxicity evaluation.
In non-rodent animals, the testis is a routinely recommended organ that needs to be weighed. The epididymis and prostate may need to be weighed depending on the specific test. In any case, the weighing value of the prostate, epididymis and other accessory sex organs of mature male animals is higher than that of immature male animals.
This is mainly because the physiology of the reproductive organs of adolescent non-rodents is very different. In addition, taking into account factors such as age, sexual maturity, and special spontaneous diseases (such as non-specific prostate hypertrophy and hyperplasia), the quality of the epididymis and prostate itself varies greatly between individual animals, which makes the interpretation of organ quality data very difficult [18] Therefore, the weighing of the epididymis and prostate organs of non-rodents is optional.
However, it is worth noting that the changes in the quality of prostate and epididymis in non-rodents caused by toxicity may also be reflected in changes in other parameters, such as changes in microscopic tissue structure or changes in testicular quality. In short, if the quality of these organs is collected, then the evaluation of the quality of immature animal reproductive organs needs to be extra cautious. In rodents and non-rodents, the normal reproductive cycle and different ages make the quality of the uterus and ovaries vary greatly between individuals.
The toxic effect of the female reproductive system is to be able to detect morphological changes through microscopy. In addition, the interpretation of the quality of the reproductive system organs needs to consider factors such as stress state and significant decline in body weight, because the change in the quality of the reproductive organs may be a secondary change caused by processing factors rather than a direct toxic effect of the test product. If the ovaries of rodents need to be weighed, STP recommends that the ovaries of rodent repeated toxicity studies less than 6 months can be weighed. The quality of the reproductive organs of female rodents is more meaningful in short-term toxicity studies (less than 6 months), because the senescence of the reproductive system of mature rats starts at 6-month-old rats.

2.5 Weighing of immune organs
STP recommends that the quality of the thymus and spleen should be evaluated in all GLP rodent toxicity studies over 7 days (except carcinogenicity studies). The changes in the quality of the thymus and spleen are sensitive indicators of immunotoxicity (immune stimulation or suppression, stress and physiological fluctuations. In addition, the pathological changes of the spleen and thymus tissues are closely related to the changes in organ quality.
However, in non-rodent toxicity studies, especially studies over 3 months, the quality of thymus needs to consider spontaneous thymic degeneration, which will complicate the interpretation of thymus quality. In non-rodent animals, the quality of the spleen may be affected by the method of anesthesia and the degree of bloodletting. The inspection and analysis of the quality of the thymus and spleen of non-rodents should follow the principle of case-by-case. If the test product may have immunotoxicity, such as monoclonal antibodies or biological agents, the spleen and thymus should be evaluated for organ quality.
If it is unclear whether changes in the quality of the spleen or thymus of non-rodents are related to processing factors, then these changes in quality may need to be ignored because the interpretation of these data is interfered with by age. Changes in the quality of the thymus and spleen need to be analyzed and explained in close connection with the changes in histopathology, because the quality of the lymphoid organs has a wide range of physiological variations. Studies have shown that the absolute quality of cynomolgus monkey spleen has a very large variation range, and even the difference between the maximum and minimum is 10.7 times, and has no correlation with animal maturity. Therefore, the evaluation of spleen quality of non-human primates needs to consider the animal’s Physiological variation.
In short, if the changes in lymphoid organ quality lack corresponding histopathological changes, then the interpretation of the quality changes must be cautious. Weighing of lymph nodes is not recommended, especially mesenteric lymph nodes, because their quality varies greatly among animals, and these tissues are difficult to separate from surrounding tissues.

3.Weighing of organs in acute toxicity studies and carcinogenicity studies

STP believes that in a toxicity study of more than 7 days, animals are systematically exposed to compounds, medical devices or special treatment conditions, and the weighing of organs has a certain evaluation value. However, in single-dose acute toxicity studies or dose escalation studies, organ weighing is of little significance, so organ weighing inspection is not recommended.
STP believes that carcinogenicity studies are not suitable for organ weighing, including carcinogenicity studies using transgenic animal models. In these long-term studies, physiological differences between normal ages and spontaneous diseases make the differences between animals larger, which interferes with the interpretation of the results of organ weighing. For example, spontaneous tumors, toxic effects or cachexia caused by tumors, unhealthy obesity, chronic kidney disease, cardiomyopathy, endocrine disorders and amyloidosis (mice), etc., these spontaneous diseases may interfere with the measurement of organ quality As a result, it affects the interpretation and analysis of the final data.

4.The focus of organ quality data interpretation

Usually the organ quality of the treatment group can be compared with the organ quality data of the control group weighed at the same time. However, if the organ quality of the control group is abnormally high or low, or the difference between the animals in this group is large, it may be more meaningful to compare the organ quality of the treatment group with the organ quality data of the background control group. However, it is necessary to consider the age, sex, body weight, feeding status, and solvent used in the background control group to be as consistent as possible with the treatment group [24].
In the study of non-rodents, because the number of animals in each group is small, the comparison with the organ quality data of the background control group is also very meaningful. The organ quality data of the background control group should be updated regularly in time to reduce the deviation caused by the different organ quality collected in a certain period of time. It is generally considered that the data in the most recent period (less than 5 years) is more appropriate than the old data.
STP recommends routine use of organ to body mass ratio for calculation and evaluation. The ratio of organ to body mass is very useful to clarify the changes in organ mass caused by processing factors, especially the individual values of organ mass and body mass of non-rodents vary greatly. The ratio of organ mass to body mass can reduce changes in organ mass caused by differences in body mass. At this time, the ratio of organ quality to brain quality is very meaningful, because the test product may affect body weight but usually does not have a significant effect on brain quality. The ratio of organ to brain mass is helpful to reduce the difference between animals, especially in non-rodent animal studies. Studies have shown that the organ-to-brain ratio is more appropriate in the evaluation of ovarian and adrenal organ toxicity, while the organ-to-body mass ratio is more appropriate in the evaluation of liver and thyroid.
If the change of organ quality is related to processing factors, then the interpretation of organ quality data should be combined with gross pathology, clinical pathology and histopathology data for an overall explanatory analysis. In the absence of gross pathological changes or histopathological changes, the interpretation of changes in organ quality needs to be very cautious. In short, the changes in organ quality found in the treatment group may not necessarily be related to treatment factors, but may only be secondary effects caused by treatment factors.
STP believes that the person in charge of pathology topics should be responsible for the evaluation of organ quality data, because usually organ quality is closely related to changes in gross pathology and histopathology, which is beneficial for toxic pathologists to contact all data for overall analysis. Therefore, before the histopathological evaluation, the person in charge of the pathology topic should check the quality of all organs and gross pathological data in advance, so as to better determine and describe the histopathological changes caused by any processing factors.

5.Conclusion

The change of organ quality is a sensitive indicator of the changes in tissues and organs induced by the compound. In the toxicology test, routinely comparing the organ quality between the treatment group and the control group can find the toxic effects caused by the test product. However, the value of organ quality assessment, the description of organ quality data and statistical analysis are the main points of concern for toxicology pathology and toxicologists.
The weighing organs stipulated in the technical guidelines for repeated dosing toxicity testing issued by the National Drug Evaluation Center are basically consistent with the main relevant guidelines of the United States and Europe. However, compared with Japan, there are some differences in requirements for the lungs, pituitary gland, epididymis, prostate, seminal vesicle glands, and salivary glands. The most fundamental principle should be to follow the principle of specific test and specific analysis [3]. For example, in respiratory research, the weighing of lung organs should be a valuable indicator. However, if it is not through the toxicity study of inhaled administration, it is considered that the evaluation of lung quality is of little significance for microscopy, and the weighing of lungs can be selective.
For another example, the quality of the cecum may have some value in antibiotics and nutrition research, but these changes are adaptive changes, not direct evidence of toxic reactions like changes in general and microscopic examinations, so they can be selectively checked. . In short, no matter what kind of research or organ evaluation, the change of organ quality should be comprehensively evaluated and analyzed based on the background of the type of test product, the mechanism of action and all other data of the test.