166
10.5.2 Tools for Male Reproductive Toxicity Evaluation
For all molecules concerned by REACH, it will be necessary to perform a battery of
physicochemical, ecotoxicity and toxicity tests. The animal testing required for
such tests could result in the use of several million animals, which is difficult to
justify. Although REACH and Directive 86/609/EEC2 encourage the use of alternative methods such as cellular models, the European Commission does not define
exactly which alternative tests should be used. The assessment is therefore based on
tests specified by the OECD, where animal testing is the rule since a few alternative
methods have been validated by this organization.
There are several possible routes by which spermatogenesis can be affected by
toxicants [19, 20]: (i) an alteration of the hypothalamic-pituitary axis; (ii) the alteration of other functions than that of the hypothalamus, the pituitary or the reproductive system, but which will secondarily lead to adverse effects on spermatogenesis;
(iii) the direct disruption of the testicular vasculature or somatic cells in the testis
(i.e., Leydig, peritubular, and Sertoli cells); and (iv) a direct effect on the germ cell
lineage. Indeed, many of the reproductive toxicants have a primary effect on the
testis, which could overshadow the effects downstream on the efferent ducts and/or
the epididymis (for a review reference [51]).
For evaluating the testicular toxicity of a substance, in vivo animal models are
rare and not always appropriate for mechanistic studies. In vitro studies carried out
so far relied on primary cultures of rodent testicular somatic cells (i.e., Leydig or
Sertoli cells) or of representative cell lines. However, such systems are not fully
relevant to study the effects of potentially toxic compounds on spermatogenesis.
Few studies have also used freshly isolated germ cells, grown alone on a short-term
period or in the presence of Sertoli cells. But the short survival of germ cells in vitro
greatly reduces the usefulness of these models (for a review see reference [52]).
Germ cell-Sertoli cell cocultures in bicameral chambers have been developed by
Weiss and coworkers [53]. These sophisticated systems reproduce the inter-Sertoli
cells tight junctions that are the main physical counterpart of the blood-testis barrier.
It thus makes it possible to study the effects and mechanism of action of reproductive toxicants on spermatogenesis, while reducing the number of animals
required [53].
Effects of reproductive toxicants on the epididymis will depend upon the dose
and time response. The design of specific experiments will be necessary to differentiate testosterone-dependent effects arising within the testis from direct effects on
the epididymis and spermatozoa. As a matter of fact, from a toxicologic point of
view the epididymis is inherently complicated as the organ structure and function
can be altered, both indirectly and directly.
The most spectacular alteration of the epididymis has been described subsequently to a complete androgen withdrawal subsequent to exposure to Ethane
dimethanesulphonate (EDS) a potent Leydig cell toxicant [54]. Androgen withdrawal rapidly leads to apoptosis of epithelial epididymal cells and to a reduction of
the epididymal tube diameter in the different regions of the organ. Any chemical
C. Pineau
10.5.2 Tools for Male Reproductive Toxicity Evaluation
For all molecules concerned by REACH, it will be necessary to perform a battery of
physicochemical, ecotoxicity and toxicity tests. The animal testing required for
such tests could result in the use of several million animals, which is difficult to
justify. Although REACH and Directive 86/609/EEC2 encourage the use of alternative methods such as cellular models, the European Commission does not define
exactly which alternative tests should be used. The assessment is therefore based on
tests specified by the OECD, where animal testing is the rule since a few alternative
methods have been validated by this organization.
There are several possible routes by which spermatogenesis can be affected by
toxicants [19, 20]: (i) an alteration of the hypothalamic-pituitary axis; (ii) the alteration of other functions than that of the hypothalamus, the pituitary or the reproductive system, but which will secondarily lead to adverse effects on spermatogenesis;
(iii) the direct disruption of the testicular vasculature or somatic cells in the testis
(i.e., Leydig, peritubular, and Sertoli cells); and (iv) a direct effect on the germ cell
lineage. Indeed, many of the reproductive toxicants have a primary effect on the
testis, which could overshadow the effects downstream on the efferent ducts and/or
the epididymis (for a review reference [51]).
For evaluating the testicular toxicity of a substance, in vivo animal models are
rare and not always appropriate for mechanistic studies. In vitro studies carried out
so far relied on primary cultures of rodent testicular somatic cells (i.e., Leydig or
Sertoli cells) or of representative cell lines. However, such systems are not fully
relevant to study the effects of potentially toxic compounds on spermatogenesis.
Few studies have also used freshly isolated germ cells, grown alone on a short-term
period or in the presence of Sertoli cells. But the short survival of germ cells in vitro
greatly reduces the usefulness of these models (for a review see reference [52]).
Germ cell-Sertoli cell cocultures in bicameral chambers have been developed by
Weiss and coworkers [53]. These sophisticated systems reproduce the inter-Sertoli
cells tight junctions that are the main physical counterpart of the blood-testis barrier.
It thus makes it possible to study the effects and mechanism of action of reproductive toxicants on spermatogenesis, while reducing the number of animals
required [53].
Effects of reproductive toxicants on the epididymis will depend upon the dose
and time response. The design of specific experiments will be necessary to differentiate testosterone-dependent effects arising within the testis from direct effects on
the epididymis and spermatozoa. As a matter of fact, from a toxicologic point of
view the epididymis is inherently complicated as the organ structure and function
can be altered, both indirectly and directly.
The most spectacular alteration of the epididymis has been described subsequently to a complete androgen withdrawal subsequent to exposure to Ethane
dimethanesulphonate (EDS) a potent Leydig cell toxicant [54]. Androgen withdrawal rapidly leads to apoptosis of epithelial epididymal cells and to a reduction of
the epididymal tube diameter in the different regions of the organ. Any chemical
C. Pineau