动物体内接种法是一种常用的检测外源病毒因子的方法，通过将外源病毒因子注入动物体内，观察动物的免疫反应来判断该病毒因子是否存在。动物体内接种法可以模拟实际的感染过程，更加贴近真实情况。体内动物接种法最常用鸡胚、小鼠和豚鼠等动物用于样品接种，上篇推文我们介绍了生物制品中国申报时动物体内接种法的最新法规要求，本篇文章我们将详细介绍美欧法规依据及差异分析。

一 法规依据

（一）Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications

此法规是美国食品药品监督管理局（FDA）发布的行业指南，旨在规范用于病毒疫苗生产的细胞基质及其他生物起始材料的表征和资质要求。

1. In Vivo Tests 

In vivo adventitious agent testing for vaccines includes inoculation of adult and suckling mice and inoculation of embryonated chicken eggs. Animal antibodyproduction tests may be performed when the potential for exposure to rodent viruses exists (e.g., through exposure to rodents or rodent cells). These tests also may be performed in other species to detect species-specific viruses. For example, inoculation of rabbits or guinea pigs may also be used to detect additional agents, when appropriate. The described animal studies must be performed in accordance with Good Laboratory Practices (GLP) regulations (21 CFR Part 58). 

针对动物体内接种，法规明确了对动物的要求，如鸡胚、乳鼠、成年小鼠、家兔等。并对动物数量、接种方式、接种剂量、观察周期等进行了描述。

a. Adult Mice 成年小鼠

This test detects adventitious viruses including lymphocytic choriomeningitis virus (LCMV), coxsackieviruses, flaviviruses, and rabies virus. Each of at least 20 adult mice weighing 15-20 grams should be inoculated intraperitoneally with 0.5 mL and intracerebrally with 0.03 mL of the material to be tested. The mice should be observed daily for 21 days. Each mouse that dies after the first 24 hours of the test, or is sacrificed because of illness, should be necropsied and examined for evidence of viral infection by gross observation and by intraperitoneal and intracerebral inoculation of appropriate homogenized tissue into at least five additional mice. Each mouse inoculated with the homogenized tissue should then be observed daily for 21 days. The material may be used only if at least 80% of the originally inoculated mice and at least 80% of each subsequently inoculated group of mice remain healthy and survive the observation period, and if none of the mice show evidence of a transmissible agent or other viral infection, other than agents known to be a component of the tested material (i.e., vaccine strains of virus, when relevant) (Ref. 12). 

b. Suckling Mice 乳鼠

This test detects adventitious agents including many human viruses, such as coxsackievirus types A and B (type B is also detectable in cell culture) and other picornaviruses (e.g., polioviruses and echoviruses), alphaviruses, bunyaviruses (e.g., phleboviruses and nairoviruses), arenaviruses, flaviviruses, rabies, and herpesviruses (e.g., herpes simplex virus).

This test can also detect many murine agents. 

Each of at least 20 suckling mice less than 24 hours old should be inoculated intraperitoneally with 0.1 mL and intracerebrally with 0.01 mL of the material to be tested. The mice should be observed daily for at least 14 days. Each mouse that dies after the first 24 hours of the test, or is sacrificed because of illness, should be necropsied and examined for evidence of viral infection by gross observation and intraperitoneal and intracerebral inoculation of appropriate tissue into at least five additional mice, which should each be observed daily for 14 days. In addition, a blind passage (via intraperitoneal and intracerebral inoculation into at least 5 additional mice) should be made of a single pool of the emulsified tissue (minus skin and viscera) of all mice surviving the original 14-day test. The material may be used only if at least 80% of the originally inoculated mice and at least 80% of each group of subsequently inoculated mice remain healthy and survive the entire observation period and if none of the mice show evidence of a transmissible agent or other viral infection, other than agents known to be a component of the tested material (i.e., vaccine strains of virus, when relevant) (Ref. 12). 

c. Guinea Pigs 豚鼠

This test detects Mycobacterium tuberculosis and adventitious viruses including paramyxoviruses (including Sendai virus), reoviruses, and filoviruses.

Each of at least 5 guinea pigs each weighing 350-450 grams should be inoculated intraperitoneally with 5 mL and intracerebrally with 0.1 mL of each material to be tested. The animals should be observed daily for at least 42 days. Each animal that dies after the first 24 hours of the test, or is sacrificed because of illness, should be necropsied. All remaining animals should be sacrificed and necropsied at the end of the observation period. The material may be used only if at least 80% of the originally inoculated animals remain healthy and survive the observation period and if none of the animals shows evidence of a transmissible agent or other viral infection, other than agents known to be a component of the tested material (i.e., vaccine strains of virus, when relevant) (Ref. 12). In vitro methods, such as culture and PCR, are also acceptable for identifying Mycobacterium tuberculosis when validated. 

d. Rabbits 家兔

This test detects simian herpes B virus, and should be considered when primary monkey cells are used. Each of at least 5 healthy rabbits each weighing 1500-2500 grams should be inoculated intradermally in multiple sites with a total of 1.0 mL of the material to be tested and subcutaneously with 2.0 mL of the material to be tested. The animals should be observed daily for at least 30 days. Each animal that dies after the first 24 hours of the test, or is sacrificed because of illness, should be necropsied. The material may be used only if at least 80% of the originally inoculated animals remain healthy and survive the observation period, and if none of the animals show evidence of a transmissible agent or other viral infection, including lesions at the site of inoculation, other than agents known to be a component of the tested material (i.e., vaccine strains of virus, when relevant) (Ref. 12). 

e. Embryonated Chicken Eggs 鸡胚

This test detects adventitious agents including: by the allantoic route: orthomyxoviruses (influenza virus) and paramyxoviruses (mumps, measles, parainfluenza viruses), alphaviruses, and vesiculoviruses; and by the yolk sac route: herpesviruses, poxviruses, rhabdoviruses, as well as rickettsiae, mycoplasmas, and bacteria. 

A sample volume, equivalent to at least 100 doses, or 10 mL, whichever represents a greater volume, should be used in egg testing. At least 10 embryonated eggs, 10 to 11 days old, should be inoculated by the allantoic route using 0.5 mL per egg. Following incubation at 35ºC for 72 hours, the allantoic fluids should be harvested, pooled, and passaged by the same route into fresh, embryonated eggs, 10 to 11 days old, using 0.5 mL per egg and incubated at 35°C for 72 hours. Both the initial pool and the passaged harvest should be tested for the presence of hemagglutinating agents with red cells from guinea pigs, humans (type O), and an avian species. The tested material passes the test if at least 80% of the embryos appear normal and there is no evidence of viral or hemagglutinating agents. 

At least 10 additional embryonated eggs, 6 to 7 days old, should be inoculated by the yolk sac route using 0.5 mL per egg. Following incubation at 35°C for at least 9 days, the yolk sacs should be harvested and pooled. A 10% suspension of yolk sacs should be passaged by the same route into the yolk sacs of fresh embryonated eggs, 6 to 7 days old, using 0.5 mL of inoculum per egg and incubated at 35°C for at least 9 days. The material passes the test if at least 80% of the embryos in both the initial test and the passage appear normal and there is no evidence of viral agents (Ref. 12).

（二）EMA(1995,Directive 75-318-EEC)

欧洲相关法规针对动物体内接种，明确了对鸡胚、乳鼠、成年小鼠、豚鼠等动物的要求，并对动物数量、接种方式、观察周期等进行了描述。

Tests in animals for adventitious agents should include the inoculation by the intramuscular route of each of the following groups of animals with the test material or with disrupted cells from the seed lot propagated beyond the maximum level (or population doubling, as appropriate) used for production:

– 2 litters of suckling mice, comprising at least 10 animals less than 24 hours old

– 10 adult mice

– 5 guinea-pigs

Test material should be injected intracerebrally into each of 10 adult mice.

The animals should be observed for at least 4 weeks. Any animals that are sick or show any abnormality should be investigated to establish the cause of illness. Test material can be considered to be suitable for production if at least 80 % of the animals inoculated remain healthy and survive the observation period and none of the animals shows evidence of the presence in the tested material of any adventitious agent.

Fertilised eggs may also act as useful substrates. Test material should be injected into eggs by appropriate routes, the chorioallantoic membrane, amniotic cavity and yolk sack of each of 10 embryonated chicken eggs, 9-11 days old. The embryonated eggs should be examined after not less than 5 days incubation. The allantoic fluids should be tested with guinea-pig and chick or other avian red cells for the presence of haemaglutinins.

（三）VIRAL SAFETY EVALUATION OF BIOTECHNOLOGY PRODUCTS DERIVED FROM CELL LINES OF HUMAN OR ANIMAL ORIGIN Q5A(R2)

ICH Q5A(R2)是国际协调会议（ICH）发布的生物技术产品病毒安全性评价指南，适用于由人或动物细胞系衍生的生物技术产品，旨在确保产品在生产过程中有效控制病毒污染风险。

The use of the in vivo assay may include inoculation of test article into suckling mice, adult mice, and embryonated hen’s eggs. The health of the animals should be monitored, and any abnormality should be investigated to establish the cause.

二 中美欧申报差异分析（以鸡胚为例）

三 总结

关于鸡胚、乳鼠和成年小鼠接种法的各国法规都存在技术细节的差异，因此申报时需要根据具体情况进行选择。恒驭生物拥有经验丰富的动物实验室团队，优秀的服务能力与平台，能够提供外源病毒检测中的动物实验操作服务，包括有小鼠试验、乳鼠试验、豚鼠试验和家兔试验。还可进行鼠源病毒抗体检测，包括小鼠鼠源病毒抗体检测、仓鼠鼠源病毒抗体检测和大鼠鼠源病毒抗体检测，以及成瘤性检测等，欢迎新老客户咨询交流！

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