This standard specifies the basic technical requirements for the mouse sperm teratology test. This standard is applicable to the evaluation of the genetic toxicity of chemical, biological and physical factors that may cause harm to health during the production, processing, storage, transportation and sales of food. The test objects include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, and microorganisms used in the food industry. GB 15193.7-2003 Mouse sperm teratology test GB15193.7-2003 standard download decompression password: www.bzxz.net

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National Standard of the People's Republic of China
GB15193.7-2003
Replaces GB15193.7-1994
Mice sperm abnormality test
Issued on September 24, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on May 1, 2004
GB 15193.7—2003
The full text of this standard is mandatory.
This standard replaces GB15193.7—1994 "Mice sperm abnormality test". Compared with GB15193.7-1994, this standard has been modified as follows: In the "Scope", the specific content of the test objects has been added: chemical, biological and physical factors involved in the production, processing, storage, transportation and sales of food that may cause harm to health; the test objects include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.; in the "Dose and Grouping": the design method of the high-dose group Added "When the animals do not die at the maximum dose (maximum concentration and maximum oral volume) of the test substance in the acute toxicity test and the LDs cannot be calculated, the following order shall be used: a) 10g/kg body weight; b) 100 times the possible human intake; or c) sub-maximum oral dose"; in "operation steps": added "test substance preparation" content; - "microscopic examination" was changed to "film reading", and the content was divided into: "film reading requirements", "types of sperm abnormalities" and "film reading precautions"; - "statistical methods and result determination" was divided into "data processing" and "result determination". From the date of implementation of this standard, GB15193.7-1994 will be abolished at the same time. This standard was proposed and managed by the Ministry of Health of the People's Republic of China. The unit of this standard: Nutrition and Food Safety Institute of China Center for Disease Control and Prevention, Zhejiang Academy of Medical Sciences. The main drafters of this standard: Xu Jinkang, Gong Youju, Chen Silan. This standard was first issued in 1994, and this is the first revision. 56
１ Scope
Mouse sperm abnormality test
This standard specifies the basic technical requirements for the mouse sperm abnormality test. GB 15193.7—2003
This standard is applicable to the evaluation of the genetic toxicity of chemical, biological and physical factors that may cause harm to health during the production, processing, storage, transportation and sales of food. The test objects include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.
2 Principle
Mouse sperm abnormality is controlled by genes and is highly heritable. Many autosomal and X and Y sex chromosome genes directly or indirectly determine sperm morphology. Sperm abnormality mainly refers to morphological abnormalities. It is known that sperm abnormality is the result of mutations in genes that determine sperm formation. Therefore, changes in morphology indicate changes in related genes and their protein products. Mouse sperm deformity test can detect the effect of environmental factors on sperm production and development, and is highly sensitive to known germ cell mutagens. Therefore, this test can be used to detect the mutagenic effect of environmental factors on germ cells in vivo.
3 Instruments and reagents
All reagents are analytically pure unless otherwise specified, and the test water is distilled water. 3.1 Common laboratory equipment.
3.2 Biological microscope.
3.3 Methanol.
3.4 ​​1%~2% eosin staining solution: weigh 1g~2g of eosin and dissolve it in 100mL of distilled water for later use. 4 Experimental animals
Adult male mice, 6 to 8 weeks old, weighing 25g~~35g. The animals were allowed to adapt to the environment for 3 to 5 days after purchase. 5 Dosage and grouping
The test substance should be set up in three dose groups. In principle, the highest dose group is the dose at which animals show severe poisoning and/or individual animals die. Generally, 1/2LDsn can be taken. The low dose group should not show toxicity, and 1/4 and 1/8LDsn are taken as the medium and low doses respectively. When the animal does not die at the maximum dose (maximum use concentration and maximum gavage capacity) of the test substance in the acute toxicity test and the LDso cannot be calculated, the high dose group is designed in the following order: a) 10g/kg body weight; b) 100 times the possible human intake, or c) - the maximum gavage dose for one time, and then the medium and low dose groups are set up. A solvent control group and a positive control group are also set up. There are at least 5 surviving animals in each group. Positive substances can be administered orally or intraperitoneally (preferably by oral administration) using cyclophosphamide 40mg/kg body weight 60mg/kg body weight, methyl methanesulfonate (MMS) 50mg/kg body weight or mitomycin C (MMC) 1.0mg/kg body weight 1.5mg/kg body weight. 6 Operation steps
6.1 Preparation of test substances
Generally, distilled water is used as the solvent. If the test substance is insoluble in water, edible oil, medical starch, carboxymethyl cellulose, etc. can be used to prepare an emulsion or suspension. The test substance should be freshly prepared before gavage, unless there is information indicating that it is stable when stored as a solution (or suspension, emulsion, etc.). 6.2 Treatment of experimental animals
GB15193.7—2003
Administer orally for 5 consecutive days. Various mutagens act on different developmental stages of sperm, and sperm deformities may occur at different times after exposure to a certain mutagen. Therefore, if conditions permit, animals can be killed at 1, 4, and 10 weeks after the test substance is administered to examine sperm morphology. Because most chemical mutagens are more sensitive to germ cells in the late spermatogonia or early primary spermatocytes, they are generally killed on the 35th day after the first administration of the test substance.
6.3 Specimen preparation
Mice are killed by cervical dislocation, and the epididymis is removed and placed in a small beaker with an appropriate amount of saline (about 1mL) or in a plate containing 2mL of saline. Use ophthalmic scissors to cut the epididymis longitudinally 1 to 2 times, let it stand for 3min to 5min, and shake it gently. Filter with four-layer lens paper or synthetic fiber blood net bag, and absorb the filtrate for smear. After air drying, fix with methanol for more than 5min, stain with 1% to 2% eosin for 1h, rinse with water, and dry.
6.4 Film reading
6.4.1 Film reading requirements
Under a low-power microscope (with a green filter), find a site with a clear background and less sperm overlap, and use a high-power microscope to sequentially check the sperm morphology and count the sperm with complete structures. Sperm with a head but no tail (unclear outline) or with a head overlapping other sperm or fragments, or those that are obviously cut up by humans, are not counted. At least 1,000 sperm are examined for each animal. 6.4.2 Types of sperm abnormalities
Sperm abnormalities are mainly manifested in the head, followed by the tail. The abnormality types can be divided into hookless, banana-shaped, fat head, amorphous, folded tail, double head, double tail, etc.
6.4.3 Notes on film reading
The coordinates of the microscope should be recorded for abnormal sperm for review. The abnormality types should also be recorded separately to calculate the sperm abnormality rate and the composition ratio of sperm abnormality types. When judging the double head and double tail deformity, it is important to distinguish it from the partial overlap of two sperms. When judging the amorphous, it is important to distinguish it from artificial cutting and folding.
7 Data processing
Each dose group should be compared with the corresponding negative control group. For example, the standard for evaluating sperm deformity positive by the Wilcoson rank sum test is that the deformity rate is at least twice that of the negative control group or is statistically significant and has a dose-response relationship. 8 Result judgment
Generally, the sperm abnormality rate of the negative control group is 0.8% to 3.4%, for reference. However, the spontaneous deformity rate of the experimental animals used in this laboratory should be used as a reference.
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