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Assessment of reproductive toxicity under REACH
Regulatory Toxicology and Pharmacology 63 (2012) 286–290
Contents lists available at SciVerse ScienceDirect
Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Commentary
Assessment of reproductive toxicity under REACH q Walter Aulmann ⇑,1 Ecolab Deutschland GmbH, Reisholzer Werftstr. 38-42, 40554 Düsseldorf, Germany
a r t i c l e
i n f o
Article history: Received 28 July 2011 Available online 28 March 2012 Keywords: Test strategy Developmental toxicity Male/female fertility Repeated dose toxicity OECD test guidelines Screening test EU chemical regulation
a b s t r a c t The European REACH regulation requires the evaluation of reproductive toxicity in screening tests according to OECD TG 421 and 422 for substances above the tonnage level of 10 tons/year. The overall aim of this paper is to increase flexibility in combination with a reduced number of experimental animals. Therefore, in contrast to the existing approach the registrant should have the possibility to file a dossier for a substance at the level of 10 tons/year and above also on the basis of data from a developmental toxicity study (OECD TG 414) plus a full-scale subacute toxicity study (OECD TG 407 according to the 1995 protocol). The proposed new test strategy takes into account overall considerations of duty of care and animal welfare. It enables an assessment of developmental toxicity on a definitive instead of a screening level. Registrants should be allowed to select between these two options, either the existing approach (OECD TG 421/407 and alternatively TG 422) or the approach proposed in this paper (OECD TG 407 plus TG 414). Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction At the level of the European Union the regulatory requirements for the assessment of industrial chemicals are laid down by the REACH legislation. Tonnage level drives the information requirements for a REACH registration. Reproductive toxicity information is needed either by testing or by other means. In the annexes VIII through X of the REACH regulation the tonnage related standard information requirements with regard to reproductive toxicity are defined: Annex VIII – annual production/import at 10 tons and more: A reproduction/developmental toxicity screening test (OECD TGs 421 or 422). Annex IX – annual production/import at 100 tons and more: A prenatal developmental toxicity study (EU B31, OECD TG 414) in one species. A study in a second species should be considered at either annex IX or at annex X level based on the outcome of the first test and all other relevant available data. A two-generation reproduction toxicity study (EU B35, OECD TG 416) in one species should be considered, if the 28-day or 90-day study indicates adverse effects on reproductive organs or tissues.
q Position paper of the Regulatory Toxicology Committee of the German Society of Toxicology. ⇑ Fax: +49 211 9893 396. E-mail address: [email protected] 1 As chairman on behalf of the GT Society Committee Regulatory Toxicology.
0273-2300/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yrtph.2012.03.003
Annex X – annual production/import at 1000 tons and more: A two-generation reproduction toxicity study (EU B35, OECD TG 416) in one species. REACH accepts the standard requirements to be adapted, either as reduced or deferred testing or as the need for extended testing (Annex XI of EC No. 1907/2006). The factors which may influence the test strategy include structural alerts, data from other toxicity studies, mutagenic and/or carcinogenic properties, data from humans exposed to the substance and human exposure patterns. In case of serious concerns certain tests with regard to reproductive toxicity may be conducted earlier than required depending on the tonnage level. On the basis of a potential reduction of animal numbers this paper recommends an alternative to the existing testing strategy, i.e. testing for OECD TG 414 prenatal developmental toxicity already at the annex VIII tonnage level as an alternative to the OECD TG 421/ 422 screening. In a testing program heading for annex IX and X tonnage levels, this will result in a remarkable reduction of use of experimental animals and at the same time provide robust and relevant data for the assessment of reproductive toxicity. Since the OECD TG 407 has been redesigned in 1995 to include the reproductive organs for histopathological evaluation, information on adverse effects on reproductive organs, predominantly in males, may also be derived from a definitive subacute toxicity study (OECD TG 407 in the version from 1995 or later). This test strategy has been common practice in the ICCA high production volume program as well as in European existing chemicals evaluation programs. The approach makes use of the 1995 re-design of the OECD
W. Aulmann / Regulatory Toxicology and Pharmacology 63 (2012) 286–290
TG 407 protocol which prescribes to increase the number of organs to be examined, including the reproductive organs. 2. Typology of test methods In general, reproductive toxicity is differentiated into two endpoints: developmental toxicity, which is the occurrence of adverse effects to the unborn, and impairment of fertility in the parent animals. Several test methods are in place to assess effects on reproduction (OECD, 2011). Some of them have screening character, some are definitive tests. Test method/end point
Developmental toxicity
Impairment of fertility
OECD OECD OECD OECD OECD
Screening Screening (Not relevant) (Limited relevance) Definitive test
Screening Screening Screening Definitive test Not relevant
TG TG TG TG TG
421 422 407 416 414
In addition, recently the Extended One-Generation Reproductive Toxicity Study was adopted as test guideline number 443 (OECD, 2011). This method, however, is not in the scope of this position paper. 2.1. Screening tests on reproductive toxicity Due to a lack of information on reproductive toxicity for a large number of existing chemicals, in the early 1990s the OECD adopted two tests (OECD TG 421 and 422) aiming at initial hazard identification for screening purposes. Both test guidelines require ten rats/sex per dose group. This number is low compared to the definitive tests, but high considering their screening character. According to the OECD TG 421 protocol gavage treatment in females starts 2 weeks before mating and is terminated 4 days after parturition. In males gavage treatment starts 2 weeks prior to mating and should cover at least 28 days. The following parameters are measured: clinical signs, body weight development and food/water consumption, testes and epididymides weights, corpora lutea implantations and resorptions as well as the stages of spermatogenesis. After parturition each litter is examined. Pups are sexed, stillbirths recorded and testes and epididymides of the males are weighed. Full histopathology is conducted in testes, epididymides and ovaries. Usually no haematology and clinical biochemistry parameters are determined. The OECD TG 422 combined test claims to address both general systemic toxicity and reproductive toxicity. The dosing scheme is similar to the one prescribed by OECD TG 421. Males are dosed for a minimum of 4 weeks. Females are dosed for 2 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled kill. Other than in OECD TG 421 limited haematology and clinical biochemistry is performed as well as histopathological examination of a larger set of organs. Also, a functional observation battery is applied for the detection of neurological disorders. Based on the protocol design these screening tests only provide limited information on developmental toxicity or fertility effects compared to the corresponding definitive tests (OECD TG 414 and 416) for the following reasons: Late post natal and post lactational manifestations are not detectable; there is limited pre-mating dosing period in males with regard to testicular toxicity;
287
Developmental toxicity including teratogenicity is not completely covered; No visceral and skeletal examinations are performed; The statistical power is low due to the small number of animals used in each of the treatment groups; Adverse effects on sexual maturation or reproductive functions in the offspring cannot be detected because they are only detectable after weaning. The ‘‘combined test’’ (OECD TG 422) also aims at generating information on general systemic toxicity in the absence of OECD TG 407. The dosing scheme, however, renders an inappropriate physiological situation. It is generally acknowledged that the physiological status between pregnant and non-pregnant animals may be different and therefore the study design may not be suitable. Thus, in case a subchronic/subacute study is available following the 1995 OECD TG 407 protocol, an additional OECD TG 414 is preferred instead of an OECD TG 422 test. 2.2. Repeated dose toxicity studies Information on repeated dose toxicity is also required at the annex VIII tonnage level. The standard test is a test with a 28 day duration, the subacute toxicity study (OECD TG 407). A wide spectrum of parameters is recorded, including haematology, clinical biochemistry, urinalysis, body weight, food/water consumption, general clinical observations, functional observations, gross necropsy and broad histopathology. The protocol for the OECD TG 407 subacute toxicity study since 1995 puts more emphasis on additional parameters such as neurotoxicity and supplementary organs. The gonads and accessory sex organs (uterus and prostate) are preserved and subjected to further examination including the determination of the weights of the reproductive organs and a refined histopathology of the testes. Therefore, the OECD TG 407 test may also serve for screening purposes on reproductive toxicity in both genders. 2.3. Two-generation studies The two-generation reproduction toxicity study is ‘designed to provide general information concerning the effects of a test substance on the integrity and performance of the male and female reproductive systems. This includes gonadal function, the oestrus cycle, mating behaviour, conception, gestation, parturition, lactation and weaning, and the growth and development of the offspring. The study may also provide information about the effects of the test substance on neonatal morbidity, mortality, and preliminary data on prenatal and postnatal developmental toxicity and serve as a guide for subsequent tests. In addition to studying growth and development of the F1 generation, this test method is also intended to assess the integrity and performance of the male and female reproductive systems as well as growth and development of the F2 generation’ (EU, 2008). At least three dose levels and a concurrent control are used. Among the parameters investigated are oestrus cycle, sperm parameters, organ weights, body weight and food/water consumption of parent animals, and body weight gain of offspring. In the offspring stillbirths, live births, and the presence of gross anomalies, sex of pups are recorded, functional investigations are performed and anogenital distance are determined. Parental animals (P and F1) and pups with external abnormalities are subjected to gross necropsy. Histopathology in parental animals covers vagina, uterus with cervix, ovaries, testis, epididymis, seminal vesicles, prostate, and coagulating gland. For the histopathological investigations of pups with external abnormalities or clinical signs special emphasis is put on the organs of the reproductive system.
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W. Aulmann / Regulatory Toxicology and Pharmacology 63 (2012) 286–290
The two generation study generally is considered as the definitive test in the hazard assessment strategy with regard to impairment of fertility. Due to the fact that it needs a huge number of animals this test is required only at higher tonnage levels (in general above 1000 tons per year) or if triggered by evidence of adverse effects on reproductive organs or tissues in available data.
2.4. Prenatal developmental toxicity studies The prenatal developmental toxicity study according to OECD TG 414 aims at the examination of adverse effects on the developing organism. Effects may be manifested by death of the organism, structural abnormalities, altered growth and functional deficiency. Developmental toxicology was formerly often referred to as teratology. The chemical is applied throughout the entire period of gestation to the day before caesarean section. During that time clinical observations of the dams take place and body weight and food consumption are recorded. After caesarean section and necropsy dams are subjected to post-mortem examinations and the uterine contents are inspected. Foetuses are sexed and body weights determined. Each foetus is examined for skeletal and soft tissue alterations. The prenatal developmental toxicity study generally is considered as the definitive test in the hazard assessment strategy with regard to developmental toxicity. Due to the fact that it needs a huge number of animals this test is also required only at higher tonnage levels (in general above 100 tons per year). Overviews on the study protocols and the parameters detected in the various test guidelines described above are collated in Tables 1 and 2, respectively.
3. Biological sensitivity of multiple indices of reproductive function Several papers compared the statistical precision and biological sensitivity of multiple indices of reproductive function, both on the male and female rodent. A prospective collaborative study in Japan with rather similar test protocols showed the high sensitivity of histopathology and sperm toxicity (Takayama et al., 1995). The study aimed at optimising the treatment period and parameters for the detection of toxicity to male fertility. Sixteen substances were investigated with a broad variety of modes of action. Parameters analysed were organ weights, spermatogenic endpoints, mating behaviour, caesarean section findings, and histopathology. The authors concluded that histopathological findings of the testis and epididymis were the most sensitive parameters for reproductive toxicity, followed by sperm toxicity. In general, this effect occurred at much lower doses than effects on the mating behaviour. Effects could be detected with 4 weeks exposure (i.e. a subacute toxicity study design) as well as with 9 weeks exposure. This analysis revealed that a treatment-related decrease in sperm counts failed to correlate with a concomitant decrease in fertility. Low doses with reduced sperm counts did affect neither implantation nor pregnancy indices. Even a 90% decrease might not affect fertility due to the excess in sperm reserve in rodents. Obviously, sperm count reduction is a sensitive, but not necessarily biological significant parameter for reproductive toxicity (Mangelsdorf et al., 2003). The Takayama findings are partially confirmed by earlier investigations. In a limited study with only three substances, Gray et al. (1989) also found sperm toxicity to be more sensitive than fertility indices.
The analysis of 27 repeated dose studies conducted by Ulbrich and Palmer (1995) also specify histopathology and organ weight as very sensitive indices to identify toxicants affecting male fertility. Sperm analysis reflected the results obtained by histopathology and organ weight measurement. As shown above the meta-analyses of several studies demonstrates that most male reproductive toxicants can be detected with adequate histopathology of the testes. A compound without adverse effect on reproductive organ weights or on testes histopathology, especially at dose levels producing significant toxicity in other organ systems, would very likely not be detected as a male reproductive toxicant in the screening study according to OECD TG 421 and TG 422. In such cases, further investigation of male reproductive toxicity is considered to be of low priority at the present tonnage level. The histopathological parameters are also obtained in an OECD TG 407 study. They can be considered sufficiently sensitive since adverse effects on gonads proved to be detectable at lower doses than changes in fertility indices (Mangelsdorf et al., 2003). A repeated dose toxicity study including histopathology of the reproductive organs and a continuous observation of estrous cyclicity is also suitable to detect female reproductive toxicity. Since approximately 27 days are required for follicular development from medium follicles, 4 weeks are considered to be the appropriate period to detect any ovarian toxicity in rats. A comparative study of 17 substances (Sanbuissho et al., 2009) showed that this is the adequate design to identify female reproductive toxicants even with substances of different modes of action for female fertility such as hormone analogues, primordial folliculli damaging agents, metabolism imbalance inducers and endocrine imbalance inducers. In addition, the histopathological findings in the ovaries were related to adverse changes in the female fertility parameters (Sanbuissho et al., 2009). However, for 7 of the 17 substances the repeated dose toxicity studies were less sensitive as compared to female fertility studies in order to detect female reproductive toxicity with lower NOAELs than identified in the general toxicity studies. Mangelsdorf et al. (2003) suppose that this may be due to the lower sensitivity of the fixation and staining methods cited by Sanbuissho et al. (2009). Nevertheless repeated dose toxicity studies may be used for a screening of potential impairment of fertility, especially with improved fixation and staining methods. The quality of the investigations very much depends on the experience of the test laboratory. This relates specifically to the histopathological determination of oestrus cyclus changes in the female reproductive organs and the way hormone dependent organs are investigated. In the new version of the OECD TG 407 as updated in 2008 this issue has been addressed. A guidance has been developed which should be used for future repeated dose toxicity studies (published on the OECD public website). Only for older studies not following the guidance a weight of evidence approach may be needed. The hazard assessor has clearly to keep in mind that the test according to OECD TG 407 alone is not a definitive test on impairment of fertility. This would have to be covered at higher tonnage levels within the assessment of information requirements.
4. Discussion This paper proposes a modification in the hazard identification strategy at the REACH Annex VIII screening level. It exactly describes an approach which has been common practice for a long time. The practice goes back to the days when an assessment strategy for existing substances was developed in the OECD high
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W. Aulmann / Regulatory Toxicology and Pharmacology 63 (2012) 286–290 Table 1 Comparison of several standard protocols for repeated dose and reproductive toxicity screening. OECD guideline EU-test regulation (440/2008/EEC) number Number or groups Animals per group and sex Number of adult animals General clinical observationsa Functional observationsb Body weight and food/water consumption Haematology Clinical biochemistry Urinalysis Gross necropsy Histopathological examination Brain (representative regions including cerebrum, cerebellum and pons), spinal cord, peripheral nerve Stomach, small and large intestines, liver, kidneys, urinary bladder, spleen, heart Trachea and lungs Adrenals, thyroid Gonads, accessory sex organs (uterus, prostate) Lymph nodes, thymus, spleen, bone marrow
407 B7 3+1 5 40 Yes Yes Yes 1/w Yes Yes Yes Yes Yes control + high dose group Yes
421 Not included 3+1 10 80 Yes No Yes 1/w No Yes Yes Yes Yes randomly selected five animals per dose
422 Not included 3+1 10 80 Yes Yes Yes 1/w Yes Yes Yes Yes Yes randomly selected five animals per dose Yes
Yes
Yes
Yes Yes Yes Yes
Yes Yes Yes Yes
Yes
a Changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards). b Sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli), grip strength and motor activity.
production volume program. The OECD strategy was never written down in any guidance document. The screening tests following the OECD TG 421 and 422 were originally designed for an initial hazard assessment and priority setting for further testing needs. This was also in the historical context of the chemical evaluation programs from the 1990s. REACH under annex VIII has adopted the OECD TG 421/422 methods which never intended to replace definitive tests. Basically, test programs have to follow the 3R principle (refine – reduce – replace). The use of animal tests should be reduced as much as possible. Testing strategies with a high consumption of experimental animals only for screening purposes do not necessarily comply with the 3R principle. It is reasonable that the European Union has written down the common practice from the OECD HPV program for the registration and evaluation of chemicals under REACH. REACH has implemented exactly this approach for the tonnage band at or exceeding 100 tons per year. ECHA already accepts the omission of the OECD TG 421/422 screening studies if a developmental toxicity study or a two generation reproductive toxicity study is available (REACH annex VIII 8.7.1). Repeated dose toxicity studies, as a standard requirement at the annex VIII and higher tonnage level, are able to detect the majority of compounds affecting fertility. Detection is possible for chemicals with many different modes of actions, including (but not limited to) endocrine modulation. Consequently, an adequately designed repeated dose toxicity study following the 1995 OECD TG 407 test protocol is expected to provide comparable information on potential effects on fertility as an OECD TG 421/422 screening study. Together with a developmental toxicity study according to OECD TG 414 performed already at the annex VIII tonnage level the registrant should have the option to omit the OECD TG 421/422 screening tests. Indeed, there might be effects that might be overlooked with this approach. One might fail to see for example effects on pup viability within the first four days. Also, impaired parturition might be missed. Such effects would only be seen in the definitive generation studies (OECD 415 and 416). In preparation of this paper the relevance of such (putatively missed) findings was discussed with experts from industry, academia and authorities. In general these
effects were considered as rare events. However, it is acknowledged that further research is needed to elucidate their relevance. A way forward could be a meta-analysis reviewing existing reproductive toxicity studies on such effects. The balance of ‘pros’ and ‘cons’ of the two different approaches should be carefully considered: While the OECD TG 422 /421 tests may render additional information on effects with so far questionable relevance (see above), they definitively miss to gather information on skeletal and visceral effects in the fetuses. The latter are only detected in an OECD TG 414 study and are highly relevant for developmental hazard identification. Therefore, the information obtained from this combination, i.e. OECD TG 414 together with OECD TG 407, can be considered more robust and relevant for the assessment of reproductive toxicity than the information from the OECD TG 421/422 screening tests only. This approach also reduces the total number of experimental animals for a test program designed for tonnage levels higher than annex VIII. At lower (10–100 tons/year) levels the registrant should have the following options: (i) In case no data are available from a subacute (OECD TG 407) toxicity study, the OECD TG 422 (combined test) is considered sufficient, as it is with the existing approach. Alternatively according to REACH a combination of OECD TG 407 and OECD 421 is an option. (ii) In case an OECD TG 407 study is available according to the protocol of 1995 and later, the performance of a developmental toxicity study according to OECD TG 414 is recommended without prior filing of a test proposal to ECHA. This package is considered sufficient to evaluate the potential for developmental toxicity. Regarding impairment of fertility screening information is obtained from the OECD TG 407 which is comparably robust to the results from an OECD TG 421 and TG 422 screening assay. (iii) In case no data are available on repeated dose toxicity and reproductive toxicity, the registrant should be allowed to generate data according to both OECD TG 407 and OECD TG 414 tests without prior discussion with ECHA.
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Table 2 Aspects of reproductive toxicity as detected according to different test protocols and test strategies. TG 416 Offspring growth, development and viability Pregnancy length and birth outcome Histopathology of sex organs and target organs Functional evaluation of fertility Oestrus cyclicity and sperm quality Litter composition Embryonic development Foetal growth Morphological variations and malformations Foetal and pup growth and survival Number of experimental animals a
TG 414
x x x x x
TG 421/422 x x x
TG 407
x
x
x x x x
x x x x
x 2600
TG 407 + 414
x (until day 3) a
80
80a
40
120
TG 407 + 414 + 416
TG 421/422 + 407 + 414 + 416
x x x x x x x x x x
x x x x x x x x x
2720
2800
Excluding offspring.
The scientific rationale of this approach holds true for any REACH tonnage level, not only for the lower end band. However, in the 10–100 tons/year band so far no flexibility is granted to the registrant to avoid the required screening tests OECD 421/ 422, whereas at the tonnage level above 100 tons per year a testing proposal has to be submitted to ECHA which is an element of flexibility per se. So, for registrations above 100 tons per year a waiving option can be filed against the conduct of the screening tests OECD 421/422. This view is in line with a fact sheet of ECHA (2009) on information requirements that outlines the following: ‘‘ECHA considers registration dossiers for substances P100 tons per year as technically complete even if they do not contain the results of a screening study for reproductive/developmental toxicity if one of the following conditions is met: ... The dossier contains either the results of, or a testing proposal for, a prenatal developmental toxicity study. . . .’’ The proposal presented here as an alternative option for the screening of reproductive toxicity is essentially nothing new. It has been common practice for almost two decades. The option to use a screening approach which is allowed in the higher tonnage range already in the low tonnage band helps to mitigate an inconsistency in the current REACH program hierarchy. 5. Conclusion The reproductive toxicity screening assay is intended for priority setting, but not for replacement of the respective definitive tests (Reuter et al., 2003). Overall, the information obtained from a combination of OECD TG 414 together with OECD TG 407 is considered to be more robust and more relevant for the assessment of reproductive toxicity than the information obtained from the OECD TG 421/422 screening tests only. Therefore, the registrant should be allowed to select between these two options, either the existing approach (OECD TG 421/407 and alternatively 422) or the approach proposed in this paper (OECD TG 407 plus TG 414). For a test program aiming at higher than annex VIII tonnage levels this approach will increase flexibility and will reduce the overall number of experimental animals needed for the safety assessment of reproductive toxicity. Even if a NOAEL derived for impairment of female fertility may be considered less robust than the corresponding dose descriptor
from a definitive test the proposed strategy is applicable for screening purposes. Conflicts of interest No competing financial interest exists. Acknowledgments The author would like to thank Dagmar Bury (Ludwigshafen), Gerrit Ehling (Hattersheim), Dorothea Eigler (Essen), Wolfgang Hillesheim (Schwetzingen), Hartmut Höke (Weinheim), Oliver Licht (Hannover), Werner Lilienblum (Hemmingen), Klaus Schneider (Freiburg), Claudia Sehner (Wiesbaden), Volker Soballa (Essen), Gisela Stropp (Wuppertal), Winfried Steiling (Düsseldorf), Wera Teubner (Basel), Michael Werner (Bad Kreuznach) for in-depth discussions and valuable contributions. References ECHA, 2009. Information requirements for repeated dose toxicity and reproductive toxicity – substances over 100 (and 1000) tonnes. Ref.: ECHA-09-FS-05-EN, Date: 15/09/2009, Fact sheet, online available at: http://echa.europa.eu/doc/ REACH/REACH_factsheet_testing.pdf. EU (2008). Council Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Gray, L., Ostby, J., Ferrell, J., Sigmon, R., Cooper, R., Linder, R., Rehnberg, G., Goldman, J., Laskey, J., 1989. Correlation of sperm and endocrine measures with reproductive success in rodents. In: Burger, E.J. (Ed.), Progress in Clinical and Biological Research, 302, New York, pp. 193–209. Mangelsdorf, I., Buschmann, J., Orthen, B., 2003. Some aspects relating to the evaluation of the effects of chemicals on male fertility. Regul. Toxicol. Pharmacol. 37, 356–369. OECD guidelines for the testing of chemicals, Paris. 2011.. Reuter, U., Heinrich-Hirsch, B., Hellwig, J., Holzum, B., Welsch, F., 2003. Evaluation of OECD screening tests 421 (reproduction/developmental toxicity screening test) and 422 (combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test). Regul. Toxicol. Pharmacol. 38, 17–26. Sanbuissho, A., Yoshida, M., Hisada, S., Sagami, F., Kudo, S., Kumazawa, T., Ube, M., Komatsu, S., Ohno, Y., 2009. Collaborative work on evaluation of ovarian toxicity by repeated-dose and fertility studies in female rats. J. Tox. Sci. 34, Special Issue SP1-SP22. Takayama, S., Akaike, M., Kawashima, K., Takahashi, M., Kurokawa, Y., 1995. A collaborative study in Japan on optimal treatment period and parameters for detection of male fertility disorders induced by drugs in rats. Int. J. Toxicol. 14, 266–292. Ulbrich, B., Palmer, A.K., 1995. Detection of effects on male reproduction – a literature survey. J. Am. Coll. Toxicol. 14, 293–327.
Contents lists available at SciVerse ScienceDirect
Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Commentary
Assessment of reproductive toxicity under REACH q Walter Aulmann ⇑,1 Ecolab Deutschland GmbH, Reisholzer Werftstr. 38-42, 40554 Düsseldorf, Germany
a r t i c l e
i n f o
Article history: Received 28 July 2011 Available online 28 March 2012 Keywords: Test strategy Developmental toxicity Male/female fertility Repeated dose toxicity OECD test guidelines Screening test EU chemical regulation
a b s t r a c t The European REACH regulation requires the evaluation of reproductive toxicity in screening tests according to OECD TG 421 and 422 for substances above the tonnage level of 10 tons/year. The overall aim of this paper is to increase flexibility in combination with a reduced number of experimental animals. Therefore, in contrast to the existing approach the registrant should have the possibility to file a dossier for a substance at the level of 10 tons/year and above also on the basis of data from a developmental toxicity study (OECD TG 414) plus a full-scale subacute toxicity study (OECD TG 407 according to the 1995 protocol). The proposed new test strategy takes into account overall considerations of duty of care and animal welfare. It enables an assessment of developmental toxicity on a definitive instead of a screening level. Registrants should be allowed to select between these two options, either the existing approach (OECD TG 421/407 and alternatively TG 422) or the approach proposed in this paper (OECD TG 407 plus TG 414). Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction At the level of the European Union the regulatory requirements for the assessment of industrial chemicals are laid down by the REACH legislation. Tonnage level drives the information requirements for a REACH registration. Reproductive toxicity information is needed either by testing or by other means. In the annexes VIII through X of the REACH regulation the tonnage related standard information requirements with regard to reproductive toxicity are defined: Annex VIII – annual production/import at 10 tons and more: A reproduction/developmental toxicity screening test (OECD TGs 421 or 422). Annex IX – annual production/import at 100 tons and more: A prenatal developmental toxicity study (EU B31, OECD TG 414) in one species. A study in a second species should be considered at either annex IX or at annex X level based on the outcome of the first test and all other relevant available data. A two-generation reproduction toxicity study (EU B35, OECD TG 416) in one species should be considered, if the 28-day or 90-day study indicates adverse effects on reproductive organs or tissues.
q Position paper of the Regulatory Toxicology Committee of the German Society of Toxicology. ⇑ Fax: +49 211 9893 396. E-mail address: [email protected] 1 As chairman on behalf of the GT Society Committee Regulatory Toxicology.
0273-2300/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yrtph.2012.03.003
Annex X – annual production/import at 1000 tons and more: A two-generation reproduction toxicity study (EU B35, OECD TG 416) in one species. REACH accepts the standard requirements to be adapted, either as reduced or deferred testing or as the need for extended testing (Annex XI of EC No. 1907/2006). The factors which may influence the test strategy include structural alerts, data from other toxicity studies, mutagenic and/or carcinogenic properties, data from humans exposed to the substance and human exposure patterns. In case of serious concerns certain tests with regard to reproductive toxicity may be conducted earlier than required depending on the tonnage level. On the basis of a potential reduction of animal numbers this paper recommends an alternative to the existing testing strategy, i.e. testing for OECD TG 414 prenatal developmental toxicity already at the annex VIII tonnage level as an alternative to the OECD TG 421/ 422 screening. In a testing program heading for annex IX and X tonnage levels, this will result in a remarkable reduction of use of experimental animals and at the same time provide robust and relevant data for the assessment of reproductive toxicity. Since the OECD TG 407 has been redesigned in 1995 to include the reproductive organs for histopathological evaluation, information on adverse effects on reproductive organs, predominantly in males, may also be derived from a definitive subacute toxicity study (OECD TG 407 in the version from 1995 or later). This test strategy has been common practice in the ICCA high production volume program as well as in European existing chemicals evaluation programs. The approach makes use of the 1995 re-design of the OECD
W. Aulmann / Regulatory Toxicology and Pharmacology 63 (2012) 286–290
TG 407 protocol which prescribes to increase the number of organs to be examined, including the reproductive organs. 2. Typology of test methods In general, reproductive toxicity is differentiated into two endpoints: developmental toxicity, which is the occurrence of adverse effects to the unborn, and impairment of fertility in the parent animals. Several test methods are in place to assess effects on reproduction (OECD, 2011). Some of them have screening character, some are definitive tests. Test method/end point
Developmental toxicity
Impairment of fertility
OECD OECD OECD OECD OECD
Screening Screening (Not relevant) (Limited relevance) Definitive test
Screening Screening Screening Definitive test Not relevant
TG TG TG TG TG
421 422 407 416 414
In addition, recently the Extended One-Generation Reproductive Toxicity Study was adopted as test guideline number 443 (OECD, 2011). This method, however, is not in the scope of this position paper. 2.1. Screening tests on reproductive toxicity Due to a lack of information on reproductive toxicity for a large number of existing chemicals, in the early 1990s the OECD adopted two tests (OECD TG 421 and 422) aiming at initial hazard identification for screening purposes. Both test guidelines require ten rats/sex per dose group. This number is low compared to the definitive tests, but high considering their screening character. According to the OECD TG 421 protocol gavage treatment in females starts 2 weeks before mating and is terminated 4 days after parturition. In males gavage treatment starts 2 weeks prior to mating and should cover at least 28 days. The following parameters are measured: clinical signs, body weight development and food/water consumption, testes and epididymides weights, corpora lutea implantations and resorptions as well as the stages of spermatogenesis. After parturition each litter is examined. Pups are sexed, stillbirths recorded and testes and epididymides of the males are weighed. Full histopathology is conducted in testes, epididymides and ovaries. Usually no haematology and clinical biochemistry parameters are determined. The OECD TG 422 combined test claims to address both general systemic toxicity and reproductive toxicity. The dosing scheme is similar to the one prescribed by OECD TG 421. Males are dosed for a minimum of 4 weeks. Females are dosed for 2 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled kill. Other than in OECD TG 421 limited haematology and clinical biochemistry is performed as well as histopathological examination of a larger set of organs. Also, a functional observation battery is applied for the detection of neurological disorders. Based on the protocol design these screening tests only provide limited information on developmental toxicity or fertility effects compared to the corresponding definitive tests (OECD TG 414 and 416) for the following reasons: Late post natal and post lactational manifestations are not detectable; there is limited pre-mating dosing period in males with regard to testicular toxicity;
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Developmental toxicity including teratogenicity is not completely covered; No visceral and skeletal examinations are performed; The statistical power is low due to the small number of animals used in each of the treatment groups; Adverse effects on sexual maturation or reproductive functions in the offspring cannot be detected because they are only detectable after weaning. The ‘‘combined test’’ (OECD TG 422) also aims at generating information on general systemic toxicity in the absence of OECD TG 407. The dosing scheme, however, renders an inappropriate physiological situation. It is generally acknowledged that the physiological status between pregnant and non-pregnant animals may be different and therefore the study design may not be suitable. Thus, in case a subchronic/subacute study is available following the 1995 OECD TG 407 protocol, an additional OECD TG 414 is preferred instead of an OECD TG 422 test. 2.2. Repeated dose toxicity studies Information on repeated dose toxicity is also required at the annex VIII tonnage level. The standard test is a test with a 28 day duration, the subacute toxicity study (OECD TG 407). A wide spectrum of parameters is recorded, including haematology, clinical biochemistry, urinalysis, body weight, food/water consumption, general clinical observations, functional observations, gross necropsy and broad histopathology. The protocol for the OECD TG 407 subacute toxicity study since 1995 puts more emphasis on additional parameters such as neurotoxicity and supplementary organs. The gonads and accessory sex organs (uterus and prostate) are preserved and subjected to further examination including the determination of the weights of the reproductive organs and a refined histopathology of the testes. Therefore, the OECD TG 407 test may also serve for screening purposes on reproductive toxicity in both genders. 2.3. Two-generation studies The two-generation reproduction toxicity study is ‘designed to provide general information concerning the effects of a test substance on the integrity and performance of the male and female reproductive systems. This includes gonadal function, the oestrus cycle, mating behaviour, conception, gestation, parturition, lactation and weaning, and the growth and development of the offspring. The study may also provide information about the effects of the test substance on neonatal morbidity, mortality, and preliminary data on prenatal and postnatal developmental toxicity and serve as a guide for subsequent tests. In addition to studying growth and development of the F1 generation, this test method is also intended to assess the integrity and performance of the male and female reproductive systems as well as growth and development of the F2 generation’ (EU, 2008). At least three dose levels and a concurrent control are used. Among the parameters investigated are oestrus cycle, sperm parameters, organ weights, body weight and food/water consumption of parent animals, and body weight gain of offspring. In the offspring stillbirths, live births, and the presence of gross anomalies, sex of pups are recorded, functional investigations are performed and anogenital distance are determined. Parental animals (P and F1) and pups with external abnormalities are subjected to gross necropsy. Histopathology in parental animals covers vagina, uterus with cervix, ovaries, testis, epididymis, seminal vesicles, prostate, and coagulating gland. For the histopathological investigations of pups with external abnormalities or clinical signs special emphasis is put on the organs of the reproductive system.
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The two generation study generally is considered as the definitive test in the hazard assessment strategy with regard to impairment of fertility. Due to the fact that it needs a huge number of animals this test is required only at higher tonnage levels (in general above 1000 tons per year) or if triggered by evidence of adverse effects on reproductive organs or tissues in available data.
2.4. Prenatal developmental toxicity studies The prenatal developmental toxicity study according to OECD TG 414 aims at the examination of adverse effects on the developing organism. Effects may be manifested by death of the organism, structural abnormalities, altered growth and functional deficiency. Developmental toxicology was formerly often referred to as teratology. The chemical is applied throughout the entire period of gestation to the day before caesarean section. During that time clinical observations of the dams take place and body weight and food consumption are recorded. After caesarean section and necropsy dams are subjected to post-mortem examinations and the uterine contents are inspected. Foetuses are sexed and body weights determined. Each foetus is examined for skeletal and soft tissue alterations. The prenatal developmental toxicity study generally is considered as the definitive test in the hazard assessment strategy with regard to developmental toxicity. Due to the fact that it needs a huge number of animals this test is also required only at higher tonnage levels (in general above 100 tons per year). Overviews on the study protocols and the parameters detected in the various test guidelines described above are collated in Tables 1 and 2, respectively.
3. Biological sensitivity of multiple indices of reproductive function Several papers compared the statistical precision and biological sensitivity of multiple indices of reproductive function, both on the male and female rodent. A prospective collaborative study in Japan with rather similar test protocols showed the high sensitivity of histopathology and sperm toxicity (Takayama et al., 1995). The study aimed at optimising the treatment period and parameters for the detection of toxicity to male fertility. Sixteen substances were investigated with a broad variety of modes of action. Parameters analysed were organ weights, spermatogenic endpoints, mating behaviour, caesarean section findings, and histopathology. The authors concluded that histopathological findings of the testis and epididymis were the most sensitive parameters for reproductive toxicity, followed by sperm toxicity. In general, this effect occurred at much lower doses than effects on the mating behaviour. Effects could be detected with 4 weeks exposure (i.e. a subacute toxicity study design) as well as with 9 weeks exposure. This analysis revealed that a treatment-related decrease in sperm counts failed to correlate with a concomitant decrease in fertility. Low doses with reduced sperm counts did affect neither implantation nor pregnancy indices. Even a 90% decrease might not affect fertility due to the excess in sperm reserve in rodents. Obviously, sperm count reduction is a sensitive, but not necessarily biological significant parameter for reproductive toxicity (Mangelsdorf et al., 2003). The Takayama findings are partially confirmed by earlier investigations. In a limited study with only three substances, Gray et al. (1989) also found sperm toxicity to be more sensitive than fertility indices.
The analysis of 27 repeated dose studies conducted by Ulbrich and Palmer (1995) also specify histopathology and organ weight as very sensitive indices to identify toxicants affecting male fertility. Sperm analysis reflected the results obtained by histopathology and organ weight measurement. As shown above the meta-analyses of several studies demonstrates that most male reproductive toxicants can be detected with adequate histopathology of the testes. A compound without adverse effect on reproductive organ weights or on testes histopathology, especially at dose levels producing significant toxicity in other organ systems, would very likely not be detected as a male reproductive toxicant in the screening study according to OECD TG 421 and TG 422. In such cases, further investigation of male reproductive toxicity is considered to be of low priority at the present tonnage level. The histopathological parameters are also obtained in an OECD TG 407 study. They can be considered sufficiently sensitive since adverse effects on gonads proved to be detectable at lower doses than changes in fertility indices (Mangelsdorf et al., 2003). A repeated dose toxicity study including histopathology of the reproductive organs and a continuous observation of estrous cyclicity is also suitable to detect female reproductive toxicity. Since approximately 27 days are required for follicular development from medium follicles, 4 weeks are considered to be the appropriate period to detect any ovarian toxicity in rats. A comparative study of 17 substances (Sanbuissho et al., 2009) showed that this is the adequate design to identify female reproductive toxicants even with substances of different modes of action for female fertility such as hormone analogues, primordial folliculli damaging agents, metabolism imbalance inducers and endocrine imbalance inducers. In addition, the histopathological findings in the ovaries were related to adverse changes in the female fertility parameters (Sanbuissho et al., 2009). However, for 7 of the 17 substances the repeated dose toxicity studies were less sensitive as compared to female fertility studies in order to detect female reproductive toxicity with lower NOAELs than identified in the general toxicity studies. Mangelsdorf et al. (2003) suppose that this may be due to the lower sensitivity of the fixation and staining methods cited by Sanbuissho et al. (2009). Nevertheless repeated dose toxicity studies may be used for a screening of potential impairment of fertility, especially with improved fixation and staining methods. The quality of the investigations very much depends on the experience of the test laboratory. This relates specifically to the histopathological determination of oestrus cyclus changes in the female reproductive organs and the way hormone dependent organs are investigated. In the new version of the OECD TG 407 as updated in 2008 this issue has been addressed. A guidance has been developed which should be used for future repeated dose toxicity studies (published on the OECD public website). Only for older studies not following the guidance a weight of evidence approach may be needed. The hazard assessor has clearly to keep in mind that the test according to OECD TG 407 alone is not a definitive test on impairment of fertility. This would have to be covered at higher tonnage levels within the assessment of information requirements.
4. Discussion This paper proposes a modification in the hazard identification strategy at the REACH Annex VIII screening level. It exactly describes an approach which has been common practice for a long time. The practice goes back to the days when an assessment strategy for existing substances was developed in the OECD high
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W. Aulmann / Regulatory Toxicology and Pharmacology 63 (2012) 286–290 Table 1 Comparison of several standard protocols for repeated dose and reproductive toxicity screening. OECD guideline EU-test regulation (440/2008/EEC) number Number or groups Animals per group and sex Number of adult animals General clinical observationsa Functional observationsb Body weight and food/water consumption Haematology Clinical biochemistry Urinalysis Gross necropsy Histopathological examination Brain (representative regions including cerebrum, cerebellum and pons), spinal cord, peripheral nerve Stomach, small and large intestines, liver, kidneys, urinary bladder, spleen, heart Trachea and lungs Adrenals, thyroid Gonads, accessory sex organs (uterus, prostate) Lymph nodes, thymus, spleen, bone marrow
407 B7 3+1 5 40 Yes Yes Yes 1/w Yes Yes Yes Yes Yes control + high dose group Yes
421 Not included 3+1 10 80 Yes No Yes 1/w No Yes Yes Yes Yes randomly selected five animals per dose
422 Not included 3+1 10 80 Yes Yes Yes 1/w Yes Yes Yes Yes Yes randomly selected five animals per dose Yes
Yes
Yes
Yes Yes Yes Yes
Yes Yes Yes Yes
Yes
a Changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards). b Sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli), grip strength and motor activity.
production volume program. The OECD strategy was never written down in any guidance document. The screening tests following the OECD TG 421 and 422 were originally designed for an initial hazard assessment and priority setting for further testing needs. This was also in the historical context of the chemical evaluation programs from the 1990s. REACH under annex VIII has adopted the OECD TG 421/422 methods which never intended to replace definitive tests. Basically, test programs have to follow the 3R principle (refine – reduce – replace). The use of animal tests should be reduced as much as possible. Testing strategies with a high consumption of experimental animals only for screening purposes do not necessarily comply with the 3R principle. It is reasonable that the European Union has written down the common practice from the OECD HPV program for the registration and evaluation of chemicals under REACH. REACH has implemented exactly this approach for the tonnage band at or exceeding 100 tons per year. ECHA already accepts the omission of the OECD TG 421/422 screening studies if a developmental toxicity study or a two generation reproductive toxicity study is available (REACH annex VIII 8.7.1). Repeated dose toxicity studies, as a standard requirement at the annex VIII and higher tonnage level, are able to detect the majority of compounds affecting fertility. Detection is possible for chemicals with many different modes of actions, including (but not limited to) endocrine modulation. Consequently, an adequately designed repeated dose toxicity study following the 1995 OECD TG 407 test protocol is expected to provide comparable information on potential effects on fertility as an OECD TG 421/422 screening study. Together with a developmental toxicity study according to OECD TG 414 performed already at the annex VIII tonnage level the registrant should have the option to omit the OECD TG 421/422 screening tests. Indeed, there might be effects that might be overlooked with this approach. One might fail to see for example effects on pup viability within the first four days. Also, impaired parturition might be missed. Such effects would only be seen in the definitive generation studies (OECD 415 and 416). In preparation of this paper the relevance of such (putatively missed) findings was discussed with experts from industry, academia and authorities. In general these
effects were considered as rare events. However, it is acknowledged that further research is needed to elucidate their relevance. A way forward could be a meta-analysis reviewing existing reproductive toxicity studies on such effects. The balance of ‘pros’ and ‘cons’ of the two different approaches should be carefully considered: While the OECD TG 422 /421 tests may render additional information on effects with so far questionable relevance (see above), they definitively miss to gather information on skeletal and visceral effects in the fetuses. The latter are only detected in an OECD TG 414 study and are highly relevant for developmental hazard identification. Therefore, the information obtained from this combination, i.e. OECD TG 414 together with OECD TG 407, can be considered more robust and relevant for the assessment of reproductive toxicity than the information from the OECD TG 421/422 screening tests only. This approach also reduces the total number of experimental animals for a test program designed for tonnage levels higher than annex VIII. At lower (10–100 tons/year) levels the registrant should have the following options: (i) In case no data are available from a subacute (OECD TG 407) toxicity study, the OECD TG 422 (combined test) is considered sufficient, as it is with the existing approach. Alternatively according to REACH a combination of OECD TG 407 and OECD 421 is an option. (ii) In case an OECD TG 407 study is available according to the protocol of 1995 and later, the performance of a developmental toxicity study according to OECD TG 414 is recommended without prior filing of a test proposal to ECHA. This package is considered sufficient to evaluate the potential for developmental toxicity. Regarding impairment of fertility screening information is obtained from the OECD TG 407 which is comparably robust to the results from an OECD TG 421 and TG 422 screening assay. (iii) In case no data are available on repeated dose toxicity and reproductive toxicity, the registrant should be allowed to generate data according to both OECD TG 407 and OECD TG 414 tests without prior discussion with ECHA.
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Table 2 Aspects of reproductive toxicity as detected according to different test protocols and test strategies. TG 416 Offspring growth, development and viability Pregnancy length and birth outcome Histopathology of sex organs and target organs Functional evaluation of fertility Oestrus cyclicity and sperm quality Litter composition Embryonic development Foetal growth Morphological variations and malformations Foetal and pup growth and survival Number of experimental animals a
TG 414
x x x x x
TG 421/422 x x x
TG 407
x
x
x x x x
x x x x
x 2600
TG 407 + 414
x (until day 3) a
80
80a
40
120
TG 407 + 414 + 416
TG 421/422 + 407 + 414 + 416
x x x x x x x x x x
x x x x x x x x x
2720
2800
Excluding offspring.
The scientific rationale of this approach holds true for any REACH tonnage level, not only for the lower end band. However, in the 10–100 tons/year band so far no flexibility is granted to the registrant to avoid the required screening tests OECD 421/ 422, whereas at the tonnage level above 100 tons per year a testing proposal has to be submitted to ECHA which is an element of flexibility per se. So, for registrations above 100 tons per year a waiving option can be filed against the conduct of the screening tests OECD 421/422. This view is in line with a fact sheet of ECHA (2009) on information requirements that outlines the following: ‘‘ECHA considers registration dossiers for substances P100 tons per year as technically complete even if they do not contain the results of a screening study for reproductive/developmental toxicity if one of the following conditions is met: ... The dossier contains either the results of, or a testing proposal for, a prenatal developmental toxicity study. . . .’’ The proposal presented here as an alternative option for the screening of reproductive toxicity is essentially nothing new. It has been common practice for almost two decades. The option to use a screening approach which is allowed in the higher tonnage range already in the low tonnage band helps to mitigate an inconsistency in the current REACH program hierarchy. 5. Conclusion The reproductive toxicity screening assay is intended for priority setting, but not for replacement of the respective definitive tests (Reuter et al., 2003). Overall, the information obtained from a combination of OECD TG 414 together with OECD TG 407 is considered to be more robust and more relevant for the assessment of reproductive toxicity than the information obtained from the OECD TG 421/422 screening tests only. Therefore, the registrant should be allowed to select between these two options, either the existing approach (OECD TG 421/407 and alternatively 422) or the approach proposed in this paper (OECD TG 407 plus TG 414). For a test program aiming at higher than annex VIII tonnage levels this approach will increase flexibility and will reduce the overall number of experimental animals needed for the safety assessment of reproductive toxicity. Even if a NOAEL derived for impairment of female fertility may be considered less robust than the corresponding dose descriptor
from a definitive test the proposed strategy is applicable for screening purposes. Conflicts of interest No competing financial interest exists. Acknowledgments The author would like to thank Dagmar Bury (Ludwigshafen), Gerrit Ehling (Hattersheim), Dorothea Eigler (Essen), Wolfgang Hillesheim (Schwetzingen), Hartmut Höke (Weinheim), Oliver Licht (Hannover), Werner Lilienblum (Hemmingen), Klaus Schneider (Freiburg), Claudia Sehner (Wiesbaden), Volker Soballa (Essen), Gisela Stropp (Wuppertal), Winfried Steiling (Düsseldorf), Wera Teubner (Basel), Michael Werner (Bad Kreuznach) for in-depth discussions and valuable contributions. References ECHA, 2009. Information requirements for repeated dose toxicity and reproductive toxicity – substances over 100 (and 1000) tonnes. Ref.: ECHA-09-FS-05-EN, Date: 15/09/2009, Fact sheet, online available at: http://echa.europa.eu/doc/ REACH/REACH_factsheet_testing.pdf. EU (2008). Council Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Gray, L., Ostby, J., Ferrell, J., Sigmon, R., Cooper, R., Linder, R., Rehnberg, G., Goldman, J., Laskey, J., 1989. Correlation of sperm and endocrine measures with reproductive success in rodents. In: Burger, E.J. (Ed.), Progress in Clinical and Biological Research, 302, New York, pp. 193–209. Mangelsdorf, I., Buschmann, J., Orthen, B., 2003. Some aspects relating to the evaluation of the effects of chemicals on male fertility. Regul. Toxicol. Pharmacol. 37, 356–369. OECD guidelines for the testing of chemicals, Paris. 2011.