OECD 231: Endocrine Disruptor Screening Program - Amphibian Metamorphosis Assay

Contáctenos

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.

AUTOR:

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

FECHA:

Enero de 2021

Introducción

‘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.

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.

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).

Reglamentaciones

In 2018, the OECD issued a revised Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals. The Conceptual Framework consists of five levels of tests and is designed to be used as a toolbox.

Level 1 – Sorting and prioritisation based on existing information.

Level 2 – In vitro assays providing mechanistic data.

Level 3 – 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 – In vivo assays providing comprehensive data on effects from endocrine and other mechanisms.

The Amphibian Metamorphosis Assay falls into Level 3 along with the Fish Short-Term Reproduction Assay, Uterotrophic Assay and Hershbergers Assay

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 – In vivo apical assays including Mammalian 2-generation toxicity, Amphibian Growth/Reproduction, Avian 2-generation, Fish 2-generation and Mysid 2-generation.

Amphibian Metamorphosis Assay

Materials and Methods

The Amphibian Metamorphosis Assay (AMA) is a screening assay intended to empirically identify substances which may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. It provides a well studied, thyroid dependent process which responds to substances active within the HPT axis, and it is the only existing assay which detects thyroid activity in an animal undergoing morphological development.

The test design involves exposing the stage 51 Xenopus laevis tadpoles to a minimum of three different concentrations of a test chemical and a water control for 21 days. Larval density is 20 tadpoles per test tank.

Day 7 Measurements

Five tadpoles are chosen at random and humanely euthanized. They are rinsed, blotted dry and body weight determined. Hind limb, snout to vent length and developmental stage are determined for each tadpole.

Day 21 Measurements

Remaining tadpoles removed from test tank and humanely euthanized. They are rinsed, blotted dry and body weight determined. Hind limb, snout to vent length and developmental stage are determined for each tadpole.

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

Histopatología

Diagnostic  criteria  include: Thyroid gland hypertrophy/atrophy Follicular cell hypertrophy Follicular cell hyperplasia

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

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


Figure 2. Thyroid glands (arrows). x10 magnification. Bar = 50 µm.


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


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

Outcomes

Effects on thyroid gland histology have been demonstrated in the absence of developmental effects. If histopathological changes are seen in the thyroid it is considered to be active. If no developmental delays and no histopathological changes are seen the chemical is considered to be thyroid inactive. This is because the thyroid gland is under the influence of Thyroid Stimulating Hormone (TSH) and any chemical which alters circulating thyroid hormone sufficiently to alter TSH secretion will result in histopathological changes to the thyroid glands.

Referencias

OECD (2018), Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption, OECD Series on Testing and Assessment, OECD Publishing, Paris. https://doi.org/10,18/9789264304741-en

OECD (2009), Test No. 231: Amphibian Metamorphosis Assay, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10,18/9789264076242-en.

Conversemos

Contáctenos