of Alternative (Non-Animal) Methods for Reproductive Toxicity Testing
6.1 Introduction: reproductive toxicity
Developmental and reproductive toxicity was not in the fore-ground of safety assessments for many years after the shock of the thalidomide disaster (Kim and Scialli, 2011) had died down. More recently, the European REACH legislation, which is extremely demanding in this field (Breithaupt, 2006; Hartung and Rovida, 2009a; Rovida and Hartung, 2009; van der Jagt et aI., 2004; Rovida, 2010), has stirred discussion again, notably because tests like the two-generation study are among the most costly and require up to 3,200 animals (two-generation study)
per substance. Another driving force is the European ban on testing for cosmetics ingredients (Hartung, 2008a). A series of activities by ECVAM, including several workshops, have tack-led this challenge and will be condensed here. The Integrated Project ReProTect (Hareng et aI., 2005) was one of its offspring, pioneering several alternative approaches.
Reproductive toxicity aims to assess possible hazard to the reproductive cycle, with certain emphasis on embryotoxicity.
Only 2-5% of birth defects can be associated with chemical and physical stress (Mattison, 20 I 0). This includes mainly the abuse of alcohol and other drugs. For the assessment of the prevalence of effects on mammalian fertility, the available database is even more limited.
This roadmap paper also has benefitted from the recent discus-sions, including the recent detailed analysis of the 2013 market-ing ban for cosmetic market-ingredient testmarket-ing in Europe (Adler et aI., 2011; Hartung et aI., 20 II; Mattison, 2010). In addition, some activities under the auspices of ILSIIHESI and the US ToxCast project have helped to clarify opportunities and challenges.
This paper will not always distinguish clearly between de-velopmental and reproductive toxicity, simply considering de-velopmental effects (teratogenicity) as the key concern within reproductive toxicity (which obviously also includes aspects of fertility and other impairments of the reproductive cycle). De-velopmental processes are especially difficult to assess
(Knud-Developmental toxicity
Malformations
Male-mediated effects
Growth retardation
Developmental variations
Fig. 6.1: Principal manifestations in developmental toxicity (modified from Pellizzer et aI., 2005)
Embryolethality
Maternal toxicity
Function impairment
Pharmaco-kinetics
sen et al., 20 II), as the timing of processes creates windows of vulnerability, the process is especially sensitive to genetic errors and environmental disruptions, simple lesions can lead to com-plex phenotypes (and vice versa), and maternal effects can have an impact at all stages.
6.1.1 Current testing
The treatment of one or more generations of rats or rabbits with a test chemical is the most common approach for identifying chemically induced adverse effects on reproduction (Fig. 6.1).
For evaluating developmental toxicity, test guidelines were designed to detect malformations in the developing offspring, together with parameters such as growth alterations and prena-tal morprena-tality (Collins, 2006). Developmental toxicity tests are considered mainly as screening tests (especially for REACH (Rovida et aI., 2011». The shorter and less complex acreeningD tests, which combine reproductive, developmental, and (option-ally) repeated dose toxicity endpoints into a single study design, are variants.
As a result of these studies (Tab. 6.1), a No Observed Effect Level (NOEL) is determined. These data then are extrapolated from animal studies to humans. In this process, safety factors are applied. This safety factor is normally 100, i.e., I % of the dose that did not cause any adverse effects is considered safe in humans (acceptable daily intake values). The value of 100 is a common default as a safety factor (based on the assumption that 10 is an estimate of interspecies and another 10 of intra-species differences), but justifiable deviations are possible in both direc-tions.
Reproductive toxicity testing has not been developed for, nor been largely applied to, chemicals in general Dwhich is often overlooked Dbut has been used predominantly for pharmaceu-ticals and pesticides. Pharmaceutica'ls are designed for oral,
high-dosage, effect-driven use, while chemicals, if at all, will typically affect the human body in a low-dose, long-term man-ner. Therefore, adapting the risk assessment of pharmaceuticals to chemical effects might not be appropriate. Despite that, the latter approach was introduced for chemicals several decades ago, but it held true only for new chemicals at a certain produc-tion volume. Very few new chemicals, however, are produced in high enough volumes to trigger such testing. Thus, experi-ence with the predictive value and performance in general for ordinary chemicals is more than limited. So are the laboratory capacities available to carry out testing. Bremer et al. (2007) showed that in both the New Chemicals Database and the US EPA HPV database, any given reproductive toxicity test has been used for less than 3% of the notified substances (Bremer et aI., 2007a) (Fig. 6.2).
Fleischer has demonstrated the limited testing facilities and a lack of sufficient scientific/technical know-how (Fleischer, 2007): A survey including 28 major independent and corporate laboratories in Europe indicated that only II offer two-gener-ation studies with a capacity of 28 substances per year. This total suggests a capacity to carry out about 50 parallel, two-gen-eration studies in Europe, each lasting about two years. Thus, every year 25 new substances can be included. The majority of this testing capacity is employed for drugs and pesticides.
Only about three general chemicals per year have been tested in two-generation studies since the introduction of the Dangerous Substances Directive in 1981 (Fleischer, 2007). Thus, testing of hundreds or even thousands of chemicals in the context of REACH will overwhelm available test capacities. This calls for adequate prioritizing to make best use of these limited resources as well as for the use of any other means to satisfy the informa-tion requirement by way of an alternative and integrated testing strategy.
Tab. 6.1: Harmonized guidelines used in screening and testing for developmental and reproductive toxicities for EU, US EPA, and OECD
. OECD (Organisation 6f '.' OPPTS (Offic~.of . EU Method
:t
W,Economic Co~operation . Prevention,'Pesticides, and and De~eloph1ertt) .") J . Toxic Substances); US EPA
. j
"'in
i::pienatal deyelopmental toxioity TG 414 (2001) OPPTS 870.3700 B 31
1" :t" 'i' ·,:.M·.·· " . (U.S. EPA, 1998)
. ' " .. 7 t
ReprodU0!i0n and fertility effects TG 416 (2001) OPPTS 870.3800 B 35
t ."< ; (U.S. EPA, 1998)
One~genera!ion reprodudtiY~i!0xicityst(J.dy. TG 415 (1983) B 34
Reproduction/development~1 toxicity TG 421 (1995) OPPTS 870.3550
screening test " ."" .ii;.' (U.S. EPA, 2000)
Combined repeated dose toxicity study TG 422 (1996) OPPTS 870.3650
"with thereproductionl develoPmental' (U.S. EPA, 2000)
I :.$creening test .
P q,
:'
c)evelopmental neurotoxicity TG 426 (2007) OPPTS 870.6300
1· < , ' . (U.S. EPA, 1998)
Extended one-generation reproductive TG 443 (2011) toxicity study
; '"
The expense and animal use associated with reproductive tox-icity testing is questionable when considering that reproductive toxicity is most probably an event with a low frequency in the universe of industrial chemicals. An independent expert panel of industrial reproductive toxicologists has concluded that, in all likelihood, less than 5% of industrial chemicals possess prop-erties that could be harmful to the developing child. We have found, by reviewing the New Chemical Database of the ECB, that 15 two-generation studies have led to only one R60 clas-sification, whereas 58 one-generation studies have led to three classifications (Bremer et aI., 2007a).
Publically available data on reproductive toxicity are very rare. Less than 5% of dossiers in the US EPA HPV database or the EU New Chemical Database (not public) contain any data in this field (Bremer et aI., 2007a). Knudsen et aI., have analyzed available data (Knudsen et aI., 2011) in various da-tabases: OIIEH5r::::National Toxicology Program (NTP) online database,for example, provides developmental effects data on only about 3% of the listed chemicals (70 of 2,330). Other
da-Total nvmber, of subsKa~ces i:t
OECD.414"'· 41
OECD415 8
10 43
Other 144
3
tabases are similarly sparse for developmental (or reproduc-tive) effects including FDArSJ. Center for Drug Evaluation and Research publicly accessible database (16.3%; 58 of 355 listed compounds), and FDArSJ.Center for Food Safety and Nutrition database (27.2%; 312 of 1,146 listed compounds; provided in Leadscope Databases (Leadscope) (Chihae Yang and Ann Richard, personal communication; see also Singh et al., 2010).
The EPA Integrated Risk Information System (IRIS) contains comprehensive reviews for 553 environmental chemicals (as of April 2010), and identifies the most sensitive or 'critical effect' as the basis for setting safe exposure levels to protect the pub-lic health. The critical effect is the first observed effect deemed adverse that is likely to occur in the most sensitive species as the dose rate of an agent increases (IRIS, 2010). Less than 2% of 533 IRIS assessments report the critical effect for the derivation ofa noncancer reference value (i.e., a safe exposure level) as being a developmental (5 of 553) or reproductive (4 of 553) effect (http://www.epa.gov/IRISI).This may be due to other effects being more sensitive, but more likely due to a lack of
de-58 55 14 Not done Not done
III EPA HPV Program Database II1II ECB Database
<II
'"
"'
2.6 2.5
.0 2.5
"'
....
"'
"
OJ
...
0 2....
<II
.c ....
...
1.5<II
>
0
'"
<II
v 1
c::
"'
...
III .tl :l.:: 0.5
0
*
o0
OECD 414 OECD 415 OECD 416 OECD 421/422
Guidelines followed
Fig. 6.2: Summary of existing data from the US-EPA HPV database and the ECB database fulfilling the standard information requirements of REACH
(modified from Bremer et aI., 2007a)
velopmental and/or reproductive effects data, which contributed to an increased uncertainty in the database for the choice of the critical effect, and resulted in a lower reference value in 85% of the cases where an uncertainty factor for an inadequate data-base was used. Finally, in one of the largest data compilations from multiple resources to-date, EPAfSJAggregated Toxicology Resource (ACToR) identified available developmental toxicity data for less than 30% of the 9,912 chemicals in commerce or of environmental interest, out of a chemical domain of418,513 generic chemicals (Judson et al., 2009).0
It needs to be stressed that the described effects do not au-tomatically point to impaired mammalian reproduction, but only to observed histopathological effects. The prevalence of reproductive toxicity is, most probably, lower than this query demonstrates.
To overcome low sensitivity, regulatory bodies often request testing in a second species. It should be stressed that the sensi-tivity of the test design requesting two species is still unknown.
But the consequence of requesting two species is dramatic: By assuming a maximum prevalence of 5% for developmental tox-icity in the universe of industrial chemicals, and by requesting additional testing in another species in case of a negative first study, the number of animals needed for developmental toxic-ity testing is nearly doubled. Fortunately, in a 2009 amendment to REACH, the original consideration of a second species was removed, though the respective guidance for developmental screening by ECHA has not yet been adapted (Rovida et aI., 2011). In addition, a side-effect of requesting a second species that is often overlooked in the current testing practice but that will have a high impact on large testing programs, is the in-crease in the rate of false-positives, and therefore the unwanted restrictions of valuable substances (Hartung and Rovida, 2009a;
Hartung,2009a).
Many regulatory agencies have recognized the need for a transformative shift and have initiated research programs to achieve the vision and goals laid out by the NRC (Leist et aI., 2008b; NRC, 2007). These include the NIEHS NTP Roadmap for the 21st Century from 2004 (National Toxicology Pro-gram, 2004) and the FDA Critical Path Initiative (Woodcock and Woosley, 2008; Woosley and Cossman, 2007) of the same year. EPA created the National Center for Computational Toxicology (NCCT) in 2005 and launched the ToxCast re-search program in 2006; in 2009, the NRC vision was largely adopted as EPArn toxicity testing paradigm (EPA, 2009). The OECD initiated a Molecular Screening for Characterizing In-dividual Chemicals and Chemical Categories Project in 2007, published a monograph on a 2007 Workshop on Integrated Approaches for Testing and Assessment, and actively utilizes Test Guideline Committees and a QSAR Expert Group to ensure global harmonization and validation of any new ap-proaches. What is most astonishing is the fact that we see more US and international activities than European contribu-tions, though at this moment the highest demand for change is created by European legislations; efforts in the EU are mainly carried out by research consortia between academia and in-dustry, with typically only long-term perspectives for transi-tion into regulatory use.
6.1.2 Frameworkfor replacing systemic toxicity by novel approaches
This framework is presented in more detail in Chapter I. The following approaches to overcome animal testing for a given area were identified:
I. Abolition of useless tests 2. Reduction to key events
3. Negative exclusion by lack of key property 4. Optimization of existing tests
5. In silico approaches 6. Information-rich single tests 7. Integrated testing strategies (ITS)
8. Pathways of Toxicity (PoT) and Systems Toxicology The distinction between (2) and (3) was made to stress that identifying positive or negative substances for a given hazard represents different approaches with different requirements as to prediction models, statistics, etc. Note that this framework remains largely on the level of hazard identification. Dose-re-sponse considerations and quantitative extrapolation to humans are not considered.
6.2 Application of the framework to