OECD 231: Endocrine Disruptor Screening Program - Amphibian Metamorphosis Assay

作者：

Sarah Broadley, BSc (Hons), MSc, PGCE, Research Scientist IV, Specialty Pathology Services; Dr. Catherine Ross, DVM, MSc, MRCVS, Pathologist, Department of Pathology Covance Laboratories Ltd., Harrogate, UK

日期：

2021年1月

简介

‘An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations’WHO-ICPS，2002年。

Following concerns that environmental chemicals may cause adverse effects to humans and wildlife, the OECD initiated a high priority activity to revise and develop guidelines to screen and test potential endocrine disruptors. Tests include an enhancement of the 28-day repeat dose test in rodents (Test guideline 407) and the Amphibian Metamorphosis Assay (AMA Test guideline 231).

‘An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations’WHO-ICPS，2002年。

Following concerns that environmental chemicals may cause adverse effects to humans and wildlife, the OECD initiated a high priority activity to revise and develop guidelines to screen and test potential endocrine disruptors. Tests include an enhancement of the 28-day repeat dose test in rodents (Test guideline 407) and the Amphibian Metamorphosis Assay (AMA Test guideline 231).

法律

2018年，经合组织发布了一个修订的概念框架，用于检测和评估内分泌扰乱化学品。概念框架由五个级别的测试组成，旨在用作工具箱。

Level 1 –Sorting and prioritisation based on existing information.

Level 2 –In vitro assays providing mechanistic data.

三级-In vivo assays providing data about selected endocrine mechanisms and effects.

Level 4 –In vivo assays providing data about adverse effects on endocrine mechanisms.

Level 5 –在体内测定中，提供了内分泌和其他机制的综合数据。

两栖动物变态测定与鱼短期繁殖测定，子宫繁殖测定和Hershbergers测定相同

Currently, in the EU there are no statutory requirements to use specific tests for endocrine disruption unless requested to do so.

In the U.S. however, testing is mandatory and the U.S. EPA have developed a two-tiered strategy.

Tier 1 Assays –A selection of in vitro and in vivo assays. The Amphibian Metamorphosis Assay and the Fish Short-Term Reproduction Assay fall into this range of tests.

Tier 2 Assays –体内顶端测定，包括哺乳动物2代毒性，两栖生长/繁殖，AVIAN 2代，鱼2代和Mysid 2代。

两栖动物变态测定

材料和方法

两栖变态测定（AMA）是旨在经验鉴定可能干扰下丘脑 - 垂体 - 甲状腺（HPT）轴的正常功能的物质的筛选测定。它提供了一种良好的研究，甲状腺依赖性过程，响应HPT轴内有源的物质，并且是唯一的现有测定检测在经历形态发育的动物中的甲状腺活性。

The test design involves exposing the stage 51Xenopus laevis蝌蚪至少三种不同浓度的测试化学品和水控制21天。幼虫密度为每次试验箱20蝌蚪。

Day 7 Measurements

在随机和人道安乐死的情况下选择五个蝌蚪。它们被冲洗，呈衬色的干燥和体重确定。每个蝌蚪确定后肢，鼻塞通风长度和发育阶段。

21天的测量

剩余的蝌蚪从测试罐中取出并人道地安乐死。它们被冲洗，呈衬色的干燥和体重确定。每个蝌蚪确定后肢，鼻塞通风长度和发育阶段。

The tadpoles are placed in Davidson’s fixative for 48 to 72 hours either as whole samples or trimmed head samples including the lower jaw.

Five tadpoles per tank are assessed for histopathology.

Histology Preparation

Tissue Trimming– The whole head is processed to paraffin wax.

Embedding –The head is embedded either ventral to dorsal on a horizontal plane (longitudinal (LS) presentation) or with the section site oriented down (transverse (TS) presentation).

Microtomy –Five step sections are taken through the thyroids and stained with Haematoxylin and Eosin

组织病理学

诊断标准包括：甲状腺润肤症/萎缩滤泡细胞肥大滤泡细胞增生

Follicular lumen area/colloid quality and follicular cell height/shape

Figure 1. Thyroid glands (arrows), longitudinal section, x2 magnification.

图2.甲状腺（箭头）。x10放大倍数。条=50μm。

Figure 3. Thyroid glands (arrows), transverse section, x2 magnification.

Figure 4. Thyroid glands (arrows), transverse section, x10 magnification.Bar = 100 µm.

Outcomes

在没有发育效应的情况下，已经证明了对甲状腺组织学的影响。如果在甲状腺中看到组织病理学变化，则认为它被认为是活性的。如果没有看到发育延迟和没有组织病理学变化，则将化学物质被认为是甲状腺无活性。这是因为甲状腺受到甲状腺刺激激素（TSH）的影响，并且任何改变甲状腺激素的任何化学物质都会充分改变TSH分泌将导致甲状腺细胞病理学变化。

参考资料

经合组织（2018年），关于评估内分泌干扰化学品的标准测试指南，经合组织检测和评估，经合组织发布，巴黎的标准化试验指南的指导案件150。https://doi.org/10.1787/9789264304741-

OECD (2009), Test No. 231: Amphibian Metamorphosis Assay, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264076242-en.