Chemicals—Test method of uterotrophic bioassay in rodents—A short-term screening test for oestrogenic properties
Some standard content:
ICS13.300;11.100
National Standard of the People's Republic of China
GB/T28647—2012
Chemicals
Uterine weight gain test in rodents
Short-term screening test for oestrogenic properties
Chemicals-Test nethod of uterotrophic bioassay in rodents-A short-term screening test for oestrogenic propertiesIssued on 2012-07-31
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaAdministration of Standardization of the People's Republic of China
Implementation on 2012-12-01
This standard was drafted in accordance with the regulations given in GB/1.1—2009.
GB/T28647—2012
The technical contents of this standard are consistent with the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guidelines No. 440 (2007) "Rodent Uterine Weight Gain Test-Short-Term Screening Test for Estrogen Effects" (English version). The following structural and editorial changes have been made to this standard: - The "Foreword\ and Initial Considerations and Limitations" in the original text of OECD440 are used as the "Introduction" of this standard; a chapter on scope is added;
- The "Appendix 1 Definitions" in the original text of OECD440 are used as the "Terms and Definitions" of this standard; - The "Appendix 2 Conceptual Framework for Determination and Evaluation of Endocrine Disruptors" in the original text of OFCD440 are used as the "Appendix A*
This standard is proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/IC251). Drafting units of this standard: Institute of Occupational Health and Poisoning Control, Chinese Center for Disease Control and Prevention, Liaoning Provincial Institute of Occupational Disease Prevention and Control, China Chemical Economic and Technological Development Center, China Inspection and Quarantine Bureau Institute of Quarantine Science. The main drafters of this standard: Hou Fenxia, Qu Bo, Yu Xiaobing, Li Xuefei, Bai Yu, Li Xi. GB/T28647—2012
In 1998, OECD launched an action with commercial priority, namely, to revise the existing guidelines and establish new guidelines for screening and detecting potential endocrine disruptors. One of the contents of the action is to establish a test guideline for the rodent uterine weight gain test. After that, an extensive validation procedure was carried out for the rodent uterine weight gain test, including the editing of detailed background documents2 and the use of strong active reference estrogens, weakly active estrogen receptor agonists, strong active estrogen receptor antagonists, negative reference chemicals, etc. as test substances, and extensive intra-laboratory and inter-laboratory comparisons to illustrate the relevance of the test. and reproducibility [4-. This test guide 440 was developed based on experience gained in validation studies and in reference to the results of a wide range of studies on test substances that are hormone agonists. The uterine weight gain test is a short screening test that began in the 1930s [37-2a and was first designated as a screening test by an expert committee in 1962 [32.35]. It is based on the increase in uterine mass or uterine weight gain response (see 5.3) and is used to evaluate the ability of a chemical to elicit biological effects consistent with the agonist or antagonist effects of natural estrogens (e.g., 17P-diol, 17βestradiol). However, this method is used much less for detecting estrogen antagonists than for detecting estrogen agonists. The uterus responds to estrogen in two ways. The initial reaction is to absorb water. Causes an increase in uterine mass, after which further growth of uterine tissue causes an increase in its mass. The uterine responses of rats and mice are comparable. This test is an in vivo screening test, and its application can be found in the "OECD Conceptual Framework for the Determination and Evaluation of Endocrine Disruptors" (Appendix A). In this conceptual framework, the uterine weight test is located at Level 3 of the framework as an in vivo test, indicating a single endocrine mechanism of action, namely estrogen-like effects. The uterine weight test is expected to be included in a series of in vivo and in vitro tests for detecting potential endocrine disrupting effects, and ultimately for risk assessment of human health and environmental effects. When conducting validation tests for this method, the OECD used strong and weak estrogen agonists to evaluate the effectiveness of the test in detecting substances with estrogen-like effects [-]. Thus, in addition to good intra- and inter-laboratory reproducibility, the sensitivity of the test for the detection of estrogen-like substances has been well established. In the validation of the method, a negative reference substance was used that was reported to be negative in the uterine weight test as well as in the in vitro receptor binding and receptor detection assays. In addition, data outside the validation studies were evaluated that further demonstrated the specificity of the uterine weight test for screening substances with estrogen-like effects. Estrogen agonists and antagonists act as ligands for the β- and β-receptors and can activate or inhibit the transcription of the receptors, respectively. This can cause potential health hazards, including effects on reproduction and development. Therefore, there is a need to rapidly evaluate whether chemicals are likely to be estrogen agonists or estrogen agonists. Antagonist. Although the affinity of the test substance for estrogen receptors and the data on in vitro tests for estrogen receptor gene transcriptional activation can provide information, this information is only one of the factors in determining whether adverse effects are likely to occur. Other determining factors include the metabolic activation and inactivation of the chemical after entering the human body, its distribution in various tissues, and its clearance from the body; these conditions depend at least in part on the route of administration and the test substance being tested. This requires understanding the activity of the test substance in the in vivo environment, unless there is data to illustrate the absorption, distribution, metabolism and excretion characteristics of the chemical. Uterine tissue undergoes rapid growth after being stimulated by estrogen, especially in experimental rodents (whose estrus cycle lasts about 4 days). Rodents, especially rats, are widely used in dangerous The uterus of rodents is therefore an appropriate target organ for in vivo screening of estrogen agonists and antagonists. This standard is based on test methods used in OECD validation studies, the results of which have shown that the method is reliable and reproducible within and between laboratories. 5 Two methods are currently available, the ovariectomized adult female method (ovx-adult female method) and the immature non-ovariectomized method (immaturc: method). OECD validation test results show that these two methods have comparable sensitivity and reproducibility. However, immature animals are more sensitive to ovariectomized adult females because they have an intact hypothalamus-pituitary-gonadal ( The HPG axis is less specific when used in tests, but has a wider range of detection than that of ovariectomized animals because it can be used to detect substances that act on the HPG axis in addition to substances that act on the estrogen receptor. The HPG axis in humans and mice is functional at about 15 years of age. Prior to this, treatment with gonadotropin-releasing hormones and the like does not promote puberty. Before reaching puberty and vaginal opening, female animals will go through several dormancy-like sexual cycles during which vaginal opening and ovulation occur, but hormone levels fluctuate. If chemicals directly or indirectly stimulate the HGP axis, it can cause early puberty, early ovulation, and accelerated vaginal opening. In addition to substances that act on the HPG axis, if the feed does not contain estrogen substances but contains high levels of metabolizable energy, it can also stimulate animal development and accelerate vaginal opening. These substances will not cause uterine weight gain in ovariectomized adult animals because their HPG axis is not functional. Considering animal welfare, this method should preferably use immature rats, which can avoid surgery on the animals and avoid the possibility of abandoning the animals due to entering the estrous cycle (see 5.4.1). Uterine hypertrophy is not entirely caused by estrogen, that is, substances other than estrogen agonists and antagonists can also cause uterine hypertrophy. For example, relatively high doses of progesterone, testosterone, or various synthetic progestins can stimulate uterine hypertrophy [20]. Any changes in uterine hypertrophy can be confirmed by histological examination of vaginal keratinization [33]. Regardless of the inducing factor, if the result of the uterine hypertrophy test is positive, its cause should generally be further elucidated. Other data, such as in vitro test data such as estrogen receptor binding test and estrogen receptor transcriptional activation test, and in vivo test data such as female animal puberty analysis, can also be used to prove estrogen-like effects. Since the uterine hypertrophy test is an in vivo screening test, the method validation test carried out must take into account both animal welfare requirements and the strategy of conducting the test in stages. The validation test is mainly to fully demonstrate its repeatability and susceptibility to detect estrogen-like effects (an aspect that many chemicals need to pay attention to), while only a small amount of validation work has been done on its detection of anti-estrogen-like effects. Only one potent anti-estrogen was used for validation, and the data on substances with clear anti-estrogen-like effects (interference with some estrogenic activity) are very limited. Therefore, this standard is aimed at estrogenic effects, and the test method for detecting anti-estrogen-like effects is contained in a guidance document. The reproducibility and sensitivity of this method for detecting pure anti-estrogen substances will be more clearly described in the future (after the method for detecting anti-estrogen-like effects has been used routinely for some time and more anti-estrogen-like substances have been detected using this method).
It is well known that all experimental procedures involving animals should comply with local animal management standards; the requirements for animal management and handling described below are minimum standards and may be superseded by local animal management requirements. The OECD has provided guidance documents on the humane treatment of animals. As with all experiments using live animals, it should be determined whether this test is necessary before the experiment. For example, this test can be carried out in the following two situations: (1) the potential exposure is large (see Level 1 in Appendix A, Concept Framework) or the results of its estrogenic effect indicate that such an effect needs to be demonstrated by in vivo experiments. (2) The results of in vivo tests at level 4 or 5 in the conceptual framework suggest that the effects produced need to be confirmed to be related to estrogenic mechanisms, which cannot be proven by in vitro tests. iiKicaoaaikAca
Chemicals
Rodent uterine weight test
Short-term screening test for estrogenic effects
GB/T 28647—2012
This standard specifies the terms and definitions of the short-term screening test method for estrogenic effects in rodent uterine weight test for chemicals, test principles, test method description, test procedures, test data and reports, and interpretation and recognition of results. This standard applies to the short-term screening rodent uterine weight test for estrogenic effects. 2 Terms and definitions
The following terms and definitions apply to this document. 2.1
antioestrogenicity
antioestrogenic effect
the ability of a chemical to inhibit the action of 17β-estradiol (cstradial173) in a lactating animal. 2.2
date of birth
date of hirth
the 0th day after birth.
dosage
a general term for the dose, number of doses and duration of the dose. 2.4
dose
the amount of the test substance administered. For uterine weight gain tests, the dose is expressed as the mass of the test substance per unit body weight of the experimental animal per day [e.g. mg/(kg * d) l
maximumtolerabledose, MTD the maximum amount of the test substance that enters the human body without causing death of the experimental animal (expressed as DL.). 2.6
oestrogenicity
oestrogenicity
The ability of a chemical to induce an effect similar to that of estradiol179 in mammals.2.7
postnatal dayx
postnatal dayx
the number of days after birth.
Esensitivity
Sensitivity
The proportion of positive results for positive/active substances. It is a measure of the precision of the test method (the ability to correctly detect substances with different activities) and is an aspect that needs to be carefully considered when evaluating the relevance of the test method.1
GB/T 28647—2012
Specificity
The proportion of negative results for negative/inactive substances. It is a measure of the precision of the test method (the ability to correctly detect substances with different activities) and is an aspect that needs to be carefully considered when evaluating the relevance of the test method. 2.10
uterotrophic
Describes a positive effect on the growth of uterine tissue. 2. 11
Validation
A scientific process used to specify the operational requirements and detection limitations of a test method and to demonstrate the reliability and relevance of the test method for a specific test day. 3 Principle of the test
3.1 The sensitivity of the uterine weight gain test relies on an animal test system, that is, the animal's hypothalamic-pituitary-ovarian axis is non-functional, resulting in very low endogenous estrogen levels. Such a test system can ensure that the uterus maintains a low quality and the animal's response to estrogen poisoning is within the most human range. There are two estrogen-sensitive states of female rodents that meet the above requirements, namely: - immature sexual animals after weaning and before puberty; - nascent female animals that have been ovariectomized and a sufficient period of time has passed since ovariectomy for the uterine tissue to regress. 3.2 Daily oral gavage or subcutaneous injection The test substance shall be administrated by injection. At least two groups shall be set up (see 5.6.1). The dose shall be graded and each group shall be administrated with one dose. If immature animals are used, the administrated period shall be 3 consecutive days; if ovariectomized adult animals are used, the administrated period shall be at least 3 consecutive days. The animals shall be dissected approximately 24 hours after the last administrated. For estrogen agonists, it shall be evaluated whether the average uterine mass of the administrated group is statistically significantly increased compared with that of the control group. If the average uterine mass of the administrated group is significantly increased, the test result is positive. 4 Description of the test method
4.1 Selection of animal species
4.1.1 Commonly used rodent strains shall be used. For example, two strains, SD rats and Wistar rats, were used in the validation of this test. If a strain of animals is known or suspected to have poor organ reactivity, animals of this strain shall not be used. The laboratory shall state the sensitivity of the animal strain used (see 5.1).
4.1.2 Rats and mice have been routinely used in uterine weight studies. The OECD used only rats in the validation of this test because it was considered that there would be no difference between the use of rats and mice and that, in order to save resources and animals, it would be sufficient to use one strain of animals for the validation studies internationally. Rats are the preferred animal species for most reproductive and developmental toxicity studies. Given the limited data on mice and the limited use of mice as rodents in human studies, some validation studies were subsequently conducted using mice. In keeping with the original goal of saving resources and animals, the validation studies were simplified by reducing the number of chemicals and laboratories involved in the validation and not using coded samples. The results of the validation study in ovariectomized juvenile mice using this simplified approach showed good qualitative and quantitative agreement between the validation results in humans and mice. If the uterine weight study is a pilot study for a long-term study, the same animal strain and source may be used for the long-term study. The simplified method mentioned above is limited to adult mice with ovariectomized, and no data on the validation test results of immature mice are provided. Therefore, the use of immature mice for testing is not considered within the scope of this standard. GB/T 28647-20 12
4.1.3 In some cases, it is possible to use mice instead of human mice. The reason for using mice should be explained based on toxicological, pharmacokinetics and/or other data. When using mice: the test method may need to be modified, for example, because mice consume more food per unit body weight than rats, the content of phytoestrogens in mouse feed should be lower than that in rats. 4.2 Feeding conditions
4.2.1 All animal husbandry operations should comply with the requirements of the animal management standards of the laboratory location. The requirements for animal management and handling described below are only minimum standards. If there are relevant local requirements, they may be followed in accordance with local animal management requirements. The temperature of the animal room should be 22℃±3℃, and the relative humidity should be at least 30%, preferably not more than 70% (except when cleaning the animal room). The target of relative humidity control is 50%~60%. Human T lighting is used, and the daily light cycle is 12 hours of light/light each. 4.2.2 Animals should have free access to food and water. New adult animals can be kept in individual cages or in groups of up to 3 per cage. Group housing is recommended for immature animals due to their young age.
4.2.3 It is known that high levels of phytoestrogens in feed can increase the uterine mass of rodents to a level sufficient to interfere with the results of uterine weight gain tests [13-15]. If immature animals are used, high levels of phytoestrogens in feed and the energy produced by metabolism can cause animals to mature prematurely. Phytoestrogens in feeds are mainly derived from soy and larvae, and the concentration of phytoestrogens in different batches of feed varies. Body weight is an important variable because food intake is related to body weight. Therefore, animals of different species and ages may receive different doses of phytoestrogens from the same feed. For immature female rats, their food intake per unit body weight is about twice that of ovariectomized female rats. For young mice, their food intake per unit body weight is about four times that of ovariectomized female rats.
4.2.4 The results of the uterine weight gain test showed that a certain level of phytoestrogens in the feed is acceptable and does not reduce the sensitivity of the test. 3.13-1. For immature SD and Wistar rats, the reference level of phytoestrogens per gram of feed should not exceed 350 μg genistein equivalents [5. This reference also applies to young adult ovariectomized rats, which consume less food per unit body weight than immature animals. If adult ovariectomized mice or rats that are relatively sensitive to phytoestrogens are used, the level of phytoestrogens in the diet should be reduced proportionally. In addition, differences in the amount of energy produced by metabolism in different diets may lead to changes in the time of onset of puberty = 21-21. 4.2.5 Before the test, the diet should be carefully selected to avoid high levels of phytoestrogens in the diet (5.9) or high metabolic energy production, which may affect the test results [115.17,13-22.36]. It is important to ensure that the laboratory can meet the requirements of 6.1.1 and 6.1.2 in the test. In order to ensure compliance with good laboratory practice (GLP) requirements, each batch of diet in the test should be sampled for analysis of phytoestrogens when necessary (for example, when the uterine mass of the control group is higher than the historical control value or an inappropriate response to the reference estrogen 17a-ethynyl estradiol is produced). The samples should be analyzed or stored frozen at 20℃, or stored in a way that prevents the samples from decomposing before analysis. 4.2.6 Some bedding materials may contain natural estrogens or anti-estrogens (e.g., bedding made of rice core is known to affect the sexual cycle of humans and mice, showing anti-estrogenic effects). The level of phytoestrogens in the selected bedding materials should be as low as possible. 4.3 Animal preparation
Randomly assign animals without any disease or physical abnormalities to the control group and the poisoned group. The possible impact on the animals due to the placement of cages should be minimized, and the animals should have unique numbers. For immature animals, it is best to keep them in the same cage with the mother mouse or the nursing mother mouse until weaning during the pre-test observation period. For newly grown animals and immature animals kept in the same cage with the mother mouse or the nursing mother mouse, the pre-test observation period is about 5 days. For immature animals that have been weaned and have no mother mouse to follow, it may be necessary to shorten the pre-test observation period because the poisoning should start immediately after weaning (see 5.3). 3
GB/T 28647--2012
5 Test steps
5.1 Laboratory proficiency verification
5.1.1 The following two methods can be selected to verify the laboratory's capability: Regular verification, which is based on the initial benchmark positive control test (5.1.2). At least every 6 months, and each time there should be a change that may affect the test results (such as feed formulation changes, changes in personnel performing uterine dissection, changes in animal strains or suppliers, etc.), verify the responsiveness of the test system (animal model) by using an appropriate dose (according to 5, 1.2) of the reference hormone, 17a-ethinylestradiol (EE) (CAS: 57-63-6). Each test should also have a control group that is poisoned with a reference estrogen in mice with an appropriate dose. If the above test does not produce the expected results, the test conditions should be checked and modified accordingly. It is recommended that the dose of the reference estrogen used in the above two methods is about 70% to 80% of the effective dose. 5.1.2 Benchmark negative control test: Before conducting this test for the first time, the laboratory should demonstrate its ability by testing the responsiveness of the animal model. At least 4 dose groups should be set up to establish the dose-effect relationship of the response to the reference estrogen: 17α-ethinylestradiol (EE) (CAS No. 57-63-6). The increase in uterine mass should be compared with the existing historical data1. If the base positive control test produces the expected results, the test conditions should be checked and modified. 5.2 Number and conditions of animals
Each treated group and control group should include at least 6 animals (applicable to both immature and ovariectomized animal models). 5.3 Age of immature animals
For uterine weight gain tests on immature animals, the birth date of each animal should be recorded. Exposure should be initiated as early as possible to ensure that endogenous estrogen levels associated with puberty have not increased after exposure. On the other hand, there is evidence that very young animals may be less sensitive. To determine the optimal age of the animals, each laboratory should obtain information from its historical data on the dates of maturity of the animals.
In general, exposure of rats can be started immediately after early weaning on day 18 of life. Exposure of rats should ideally be completed on day 21 of life, but no later than day 25 of life, because after this time the hypothalamus-pituitary-ovarian axis of the rat begins to function, endogenous estrogen levels may begin to increase, and the mean and standard deviation of uterine mass also increase by 23.1 = 12. 5.4 Procedure for Ovariectomy
5.4.1 For ovariectomized female rats and mice (treated and control groups), ovariectomy should be performed between 6 and 8 weeks of age. For rats, a minimum of 14 days should elapse between ovariectomy and the first exposure to allow for minimal and stable uterine retraction. For mice, the minimum interval between ovariectomy and exposure to the drug is 7 days. Since a small amount of ovarian tissue is sufficient to induce significant endogenous hormone levels, vaginal epithelial smears should be performed on animals for 5 consecutive days (e.g., 10 to 14 days after ovariectomy) to detect the estrus cycle of the animals before use. If an animal is proven to be in the "estrus period", it cannot be used for the experiment. In addition, during the autopsy, attention should be paid to checking whether there are residual ovaries. Animals with residual ovarian tissue should not be counted in the results. 5.4.2 When performing ovariectomy, place the animal in a ventral recumbent position using an appropriate method. An incision should be made on the posterior and lateral wall of the abdominal cavity. The incision should be a longitudinal incision of about 1 cm in length, with the midpoint between the lower edge of the ribs and the top of the bone, and a few millimeters laterally from the lateral edge of the psoas muscle. The ovary is removed. Remove from the abdominal cavity to a sterile environment. Separate the ovary from the fallopian tube and uterine body. After confirming that there is no heavy bleeding, suture the abdominal wall and close the skin incision with clips or suture with appropriate sutures: the cut points are shown in Figure 1. Postoperative analgesia should be given to animals using methods recommended by experienced rodent veterinarians4
iiKacaOaiKAca=
All cut sites
一子坑
Do not polyamine membrane, blood vessels and fat layer
GB/T 28647—2012
Instructions: To perform an oophorectomy, first make an incision in the posterolateral abdominal wall, the midpoint between the inferior margin and the iliac bone, and a few meters laterally from the lateral edge of the psoas muscle. In a symptomatic area, the ovary is removed from the abdomen, a bandage is placed between the ovary and the uterus to stop bleeding, and the ovary is removed at the junction of the fallopian tube and each uterine horn. Make sure there is no large persistent bleeding shoulder and close the abdominal wall. The skin incision is closed with clips or with appropriate sutures. At least 14 days should be allowed for the animal to recover and the uterus to be minimized before use. Figure 1 Schematic diagram of the surgical process of removing the ovary
5.5 Body weight
For spayed animals, body weight and uterine mass do not correspond because uterine mass is affected by hormones (such as estrogen) but not by growth factors that regulate body size. However, for weaned animals that have not had their ovariectomized, body weight and uterine mass are related during sexual maturity. Therefore, for immature animals, the difference in animal weight at the beginning of the experiment should be minimal or no more than ±20% of the average body weight. This means that breeders should keep the same number of pups in each litter to ensure that each litter can receive essentially the same care. Randomly assign the animals to groups (control group and poisoned group) so that there is no statistically significant difference in the average body weight of each group. Without increasing the total number of experimental mice, try to avoid assigning mice with the same poor quality to the same poisoned group. 5.6 Dosage
5.6.1 In order to determine whether the test substance has an estrogenic effect in vivo, it is generally sufficient to set up two dose groups and one control group. This design is also most in line with animal welfare requirements. If you want to obtain a dose-response curve or to extrapolate to a lower dose, only three dose groups are needed. If you need to obtain information other than estrogenic effects (such as estimating its effect If the test substance is not exposed to the rats, other dose designs may be considered. Except for the non-exposed test substance, the control group animals should be treated the same as the exposed group. If a certain solvent is used when exposing the test substance, the control group should use the same amount of solvent as the exposed group (if the amount of solvent used in each exposed group is different, the control group should use the highest amount of solvent). 5.6.2 The dose design of the uterine enhancement test is to select a dose that can ensure the survival of the animals after 3 consecutive days of exposure to the test substance and does not cause obvious toxicity or pain to the animals. The maximum dose does not exceed 1000 mg/(kg·d). The test substance or its Existing toxicity and toxicokinetics data on the substance. The highest dose should first refer to the LD value and/or acute toxicity data of the test substance to avoid death, serious illness and painful manifestations in animals24-2. The highest dose may represent the MTD; it may also be the dose level that causes a positive uterine weight gain response. As a screening test, a large dose interval (e.g. 0.5 log unit, corresponding to a dose interval of 3.2; or larger, using 1 log unit) is generally acceptable. If there is no suitable data for reference, the dose range may be determined by conducting a preliminary study. ||tt ||5.6.3 In addition, if the in vitro test (or structure-activity) of the test substance can provide information on its estrogen-like effects, this information can also be used for dose design. For example, to understand at what dose of the test substance that causes uterine weight gain is equivalent to the dose of the reference estrogen agonist (ethinyl estradiol) that causes this effect, it can be understood by evaluating the dose equivalent to the effect of ethinyl estradiol in the in vitro test. The corresponding ethinyl estradiol dose can be multiplied by a factor, such as 10 or 100, as the highest dose of toxicity, 5
GB/T 28647--2012
5.7 Issues to consider in determining the dose range
If necessary: A pilot study with a small number of animals can be used to explore the dose range. OFCD Guidance Document No. 19 (2) can be used to determine whether the animals have clinical signs that indicate animal toxicity and distress. If the dose range determined in the pilot study is feasible, the uterus should be isolated and weighed approximately 21 hours after the first dose. Then: This information can be used as a reference when designing the formal study (selecting an acceptable maximum and lower doses and determining the number of dose groups). 5.8 Administration
5.8.1 Administration of the test substance by oral gavage or subcutaneous injection should be considered from the perspectives of animal welfare and toxicology. Toxicology needs to consider the relevance of the route of human exposure to the chemical (e.g., when humans are exposed orally, animals are exposed by gavage; when humans are exposed by inhalation or skin, animals are exposed by subcutaneous injection), the physical/chemical properties of the test substance, especially the existing physical information and metabolic kinetics (e.g., the test substance should be prevented from being metabolized at the first absorption site so that it is more efficiently absorbed through a specific route).
5. 8.2 It is recommended to consider using aqueous solutions or water suspensions as much as possible. Since most hormone ligands or their metabolic precursors are often insoluble in water, the most common method is to use oil solutions or oil suspensions (e.g., corn oil, peanut oil, sesame oil, or olive oil). These oils contain a certain amount of calories and fat, which can change the total amount of metabolizable energy ingested by the animal and potentially affect the observed endpoints of the test (e.g., uterine mass). Therefore, a test should be carried out before the test to show whether the results of the solvent control group are different from those of the control group without solvent. The test substance can be dissolved in the smallest possible amount of 95% ethanol or other appropriate solvent and diluted to the final working concentration with the solvent. The toxicity characteristics of the solvent should be understood and a solvent control group should be established during the test. If the test substance is stable, slight heating and strong mechanical action can be used to promote its dissolution. The qualitative properties of the test substance in the solvent should be determined. If the test substance is stable during the test, a stock solution of the test substance can be prepared first, and the specified dose of dilution can be prepared every day. 5.8.3 The duration of exposure depends on the animal model used (for immature animal models, see 5.3. For ovariectomized animal models, see 5.4). Immature female rats are exposed to the test substance once a day for 3 consecutive days. It is also recommended to use 3-day exposure for ovariectomized female rats, but increasing the number of exposure days is also acceptable and can help detect weak estrogenic activity test substances. For female mice with ovariectomized, 3 days of exposure should be sufficient. For strong hormone-like test substances, extending the exposure for a full 7 days is not very meaningful. However, whether the exposure of weak estrogen-like test substances needs to be extended to 7 days has not been proven during method validation. Therefore, can ovariectomized mice be exposed continuously? 1. Exposure should be conducted at basically the same time every day, and the exposure maximum should be adjusted according to the weight of the animal [e.g. m/(kgd)]. The concentration of the exposure solution should be adjusted to keep the exposure volume per unit body weight basically unchanged. Regardless of the exposure route, this can ensure that the exposure volume per unit body weight of each dose group remains constant.
5.8.4 When the exposure is conducted by gavage, the animal should be exposed once each time using a stomach tube or an appropriate intubation tube. The maximum volume of the test solution for each exposure should be determined according to the size of the experimental animal. The requirements of local animal care should be followed, but the maximum volume should not exceed 5 mL/kg. For aqueous solutions, the maximum volume is 10 mL/kg.
5.8.5 When the test substance is administered subcutaneously, the test substance should be administered once per day: the test substance should be injected into the dorsal shoulder or lumbar region using a sterile needle (e.g., 23-gauge or 25-gauge (as specified in OECDTG440) and a tuberculin syringe. Whether the injection site is hairless or not is optional. Any loss, leakage, or incomplete injection at the injection site should be recorded. The total amount of injection per rat per day should not exceed 5 mL/kg and should be injected at two sites. The total amount of aqueous solution injection can be 10 mL/kg. 5.9 Observation
5.9.1 General and clinical observations
General clinical observations should be performed at least once a day, and the number of observations per day should be increased if sexual manifestations are observed. It is best to observe at a fixed time of the day, taking into account the expected peak effect time after the administration of the drug. Observe the mortality, morbidity and clinical manifestations of all animals, such as changes in behavior, skin, hair, eyes, mucous membranes, secretions and excretions, and autonomic behavior (e.g. tearing, hair erection, changes in pupil size, abnormal breathing pattern).
5.9.2 Body weight and feed consumption
GB/T28647—2012
Before the start of the exposure, that is, after the animals are randomly grouped, all animals should be weighed once a day to the nearest 0.1g. The feed consumption during the exposure period may or may not be measured. When measuring, the animal keeper can measure the feed consumption of each cage of animals during the period. Feed consumption should be expressed as grams/rat/day. 5.9.3 Uterine dissection and weighing
5.9.3.1 Rats should be euthanized 24h after the last exposure. Ideally, the order of dissection of each group of animals should be randomly arranged to avoid minor effects caused by dissection from high-dose group to low-dose group or from low-dose group to high-dose group. This study is to measure the wet weight of the uterus and the weight of the uterus after removing the intrauterine fluid. The wet weight of the uterus includes the weight of the uterus and the intrauterine fluid. The weight of the uterus after removing the intrauterine fluid is measured after removing the intrauterine components. 5.9.3.2. In immature animals, the opening of the vagina should be checked before the uterus is removed. The dissection process begins with opening the abdominal wall from the pubic symphysis. Once the trumpet-shaped uterus and ovaries are visible, they are separated from the posterior abdominal wall. The bladder and ureters are removed from the ventral and lateral surfaces of the uterus and vagina. The fibrous adhesions between the rectum and vagina are separated until the junction of the vaginal opening and the perineal skin can be seen. The uterus and vagina are separated from the body by cutting the vaginal wall above the confluence of the perineal skin, see Figure 2. Carefully cut all mesentery connected to the trumpet-shaped uterus and separate the uterus from the posterior body wall. Once the uterus is separated from the body, it should be handled as soon as possible to prevent the tissue from drying out. For small tissues such as the uterus, the loss of mass due to drying is obvious. The ovaries should be separated at the oviduct to prevent the fluid from the uterine cavity from flowing out. For animals with ovariectomized animals, the presence of residual ovarian tissue should be checked. Excess adipose tissue and connective tissue should be removed. The vagina should be separated slightly below the cervix so that the cervix can be combined with the uterine body, see Figure 2. Uterus weighing
Weight
Gross dissectionbzxz.net
Instructions: First, cut the wall from the pubic symphysis and separate the ovaries and uterus from the posterior abdominal wall. Remove the bladder and urethra from the ventral and lateral surfaces of the uterus and vagina. Dissect the fibrous tissue between the rectum and vagina until the point where the penis opening connects to the perineal skin is visible. Separate the uterus and vagina by cutting the vaginal wall above the point where the penis and perineal skin connect. Carefully cut all mesentery connected to the uterus from the posterior and lateral abdominal cavity and separate the uterus. Remove excess fat and connective tissue from the uterus. Cut the ovaries at the oviduct and avoid the uterine cavity from flowing out of the uterus. For animals that have been oviposited, check for any residual ovarian tissue. Separate the vagina from the uterus just below the cervix so that the cervix remains with the uterus. Then, weigh the uterus. Figure 2 Schematic diagram of tissue removal and preparation for determination of uterine mass 5.9.3.3 Place each uterus in a uniquely numbered and weighed container (such as a petri dish or a plastic weighing bottle) and continue to carefully store it to prevent the tissue from drying out before weighing (such as placing a filter paper slightly moistened with saline in the container). The uterus containing the contents of the uterine cavity (wet weight of the uterus) should be weighed to an accuracy of 0.1 mg. 5.9.3.4 Then remove the fluid from each uterine cavity. Puncture or cut both uteri longitudinally. Place the uterus on slightly damp filter paper (such as Whatman No. 3) and gently squeeze with another slightly damp filter paper to completely remove the fluid from the uterine cavity. The uterine mass after the fluid in the uterine cavity is removed should be weighed to an accuracy of 0.1 mg. 7
GB/T 28647--2012
5.9.3.5 At the end of the study, the weight of the uterus can be used to demonstrate that the age of the immature model rats used is not above the appropriate age required for the study. This requires reference to the historical data of the strain of rats selected for the study (see ",3). 5.9.4 Other tests
The following tests can be selected: After weighing, the uterus is fixed in 10% neutral buffered formalin and stained with eosin for histopathological examination. The vagina can also be examined histopathologically (see paragraph 9 of the introduction). In addition, morphological measurements of the endometrial epithelium can be made for quantitative comparison.
6 Test data and report
6.1 Data
The test data should include:
... Number of animals at the beginning of the study;
Number and number of animals that died or were euthanized during the study, and the date and time of death or euthanasia; number and number of animals showing toxicity, observation 6.2 Data Analysis
The following data should be recorded for each animal: body weight, uterine uterus weight and uterine mass after removal of the uterine cavity. When analyzing whether the test substance causes a statistically significant change in uterine weight, a one-sided statistical analysis (p 0.05) should be used. Appropriate statistical analysis methods should be used to test whether the increase in uterine mass is statistically significant: for example, analysis of covariance (ANCOVA) can be used to analyze the data, using the body weight of the animals at the time of administration as a covariate. The data on uterine mass can be log-transformed for variance stability before analysis. Dunnett and Hsu's test is suitable for making pairwise comparisons between each dose group and the solvent control group, and calculating confidence intervals. Studentised residual plots can be used. residual plot) to find deviations from human values and perform variance consistency tests. OECD used the above statistical methods when performing analysis using the 8th version of the generalized linear model class (PROCGLM) statistical analysis system (SAS Institute, Cary, NC) software "6Ⅱ when validating the method. 6.3 Final report
The final report should include the following:
6.3.1 Test agency
---Personnel involved in the test and their responsibilities,
Data of the baseline positive control test and the data of the regular positive control group (see 5.1,1 and 5.1.2), 6.3.2 Test substance
Description of the test substance;
---Physical properties and related physicochemical properties; Dilution preparation potential and preparation frequency;
---Any information on stability;
Any analysis of the prepared test solution. 8
iiKacaOaaiKAca1 Data
The test data should include:
... the number of animals at the start of the test;
the number and number of animals that died or were euthanized during the test, and the date and time of death or euthanasia;
the number and number of animals that showed toxicity, and a description of the observed toxicity, including the time of onset, duration and severity;
the number and number of animals that showed any injury, and a description of the type of injury. 6.2 Data Analysis
The following data should be recorded for each animal: body weight, uterine weight and uterine mass after removal of the uterine cavity. When analyzing whether the test substance causes a statistically significant change in uterine weight, a one-sided statistical analysis (P 0.05) should be used. Appropriate statistical analysis methods should be used to test whether the increase in uterine mass is statistically significant: for example, the covariance analysis method (ANCOVA) can be used to perform statistical analysis on the data, with the body weight of the animal at the time of administration as the covariate. The data on uterine mass can be logarithmically transformed for variance stability before analysis. Dunnett and Hsu's test was suitable for pairwise comparisons of each dose group with the solvent control group and for calculation of confidence intervals. Studentised residual plots were used to detect outliers and to perform variance consistency tests. The OECD used the above statistical methods when conducting method validation using the PROCGLM statistical analysis system (SAS Institute, Cary, NC) version 8 software "6Ⅱ for analysis. 6.3 Final report
The final report should include the following:
6.3.1 Test agency
---Personnel involved in the trial and their responsibilities,
Data from the baseline positive control test and the data from the periodic positive control group (see 5.1.1 and 5.1.2), 6.3.2 Test substance
Description of the test substance;
---Physical properties and related physicochemical properties; Dilution preparation potential and preparation frequency;
---Any information on stability;
Any analysis of the prepared test substance solution. 8
iiKacaOaaiKAca1 Data
The test data should include:
... the number of animals at the start of the test;
the number and number of animals that died or were euthanized during the test, and the date and time of death or euthanasia;
the number and number of animals that showed toxicity, and a description of the observed toxicity, including the time of onset, duration and severity;
the number and number of animals that showed any injury, and a description of the type of injury. 6.2 Data Analysis
The following data should be recorded for each animal: body weight, uterine weight and uterine mass after removal of the uterine cavity. When analyzing whether the test substance causes a statistically significant change in uterine weight, a one-sided statistical analysis (P 0.05) should be used. Appropriate statistical analysis methods should be used to test whether the increase in uterine mass is statistically significant: for example, the covariance analysis method (ANCOVA) can be used to perform statistical analysis on the data, with the body weight of the animal at the time of administration as the covariate. The data on uterine mass can be logarithmically transformed for variance stability before analysis. Dunnett and Hsu's test was suitable for pairwise comparisons of each dose group with the solvent control group and for calculation of confidence intervals. Studentised residual plots were used to detect outliers and to perform variance consistency tests. The OECD used the above statistical methods when conducting method validation using the PROCGLM statistical analysis system (SAS Institute, Cary, NC) version 8 software "6Ⅱ for analysis. 6.3 Final report
The final report should include the following:
6.3.1 Test agency
---Personnel involved in the trial and their responsibilities,
Data from the baseline positive control test and the data from the periodic positive control group (see 5.1.1 and 5.1.2), 6.3.2 Test substance
Description of the test substance;
---Physical properties and related physicochemical properties; Dilution preparation potential and preparation frequency;
---Any information on stability;
Any analysis of the prepared test substance solution. 8
iiKacaOaaiKAca
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.