Type, duration and dose of regulatory studies

As said in General aspects of PCS, the following study types are needed for development and registration of new drugs (ICH M3(R2))

In vivo repeat dose toxicity studies of various durations depending upon the development stage and the intended duration of application to humans as specified in ICH M3(R2), generally in a rodent and a non-rodent species

Maximum duration of clinical trial	Recommended minimum duration of repeat-dose toxicity studies to support clinical trials
Maximum duration of clinical trial	Rodents	Non-rodents
Up to 2 weeks	2 weeks^a	2 weeks^a
2 weeks to 6 months	Same duration as clinical trials^b	Same duration as clinical trials^b
> 6 months	6 months^b	9 months^b,c,d

a) FDA: as alternative, extended single-dose toxicity studies can support single-dose human trials. Clinical studies of < 14 days: toxicity studies of the same duration
b) In some circumstances, clinical trials of > 3 months can be initiated, if 3-month data are available from 2 species and if complete data from the chronic studies (see line below) are made available before extending clinical trials > 3 months
c) If the primary population is pediatric and if potential developmental concerns have been identified, long-term toxicity testing starting in juvenile animals can be appropriate
d) In the EU, studies of 6 months duration in non-rodents are considered acceptable

Duration of intended therapy	Recommended duration of repeat-dose toxicity studies to support marketing
Duration of intended therapy	Rodents	Non-rodents
Up to 2 weeks	1 month	1 month
> 2 weeks to 1 month	3 months	3 months
> 1 month to 3 months	6 months	6 months
> 3 months	6 months^c	9 months^c,d

c) and d) as above

If feasible e.g. based on volume, the high dose (HD) should induce some toxicity, but not interfere largely with animal survival. The low dose (LD) should be devoid of toxicity and, in case of drugs, generally be a small multiple of the anticipated clinical dose. The medium dose (MD) is in between the LD and HD, often the geometric mean between LD and HD

In vivo carcinogenicity studies generally in 2 rodent species treated for their life-time (usually 2 years). See also presentation Carcinogenicity studies - Duration and dose selection
Safety pharmacology, generally of the cardiovascular system (non-rodents, ECG), behavior (rodents) and respiration (rodents, generally observation is sufficient); if stand-alone studies: generally by single application
Reproductive toxicity testing, generally for drugs with three study types:
- Segment I: Fertility and reproductive performance, generally done in one rodent species, mainly rats. This study serves to determine potential adverse effects of the test article on male and female fertility including evaluation of estrous cycles and sperm analysis as well as on early embryonic development. Parental males and females are administered the test article prior to mating, during the mating period and through gestation day 6 (females) or until terminal sacrifice (males; often after > 4 weeks of treatment). Thus, this is the only study for examining the effect of the test article on the sexual function of males
- Segment II: Teratology studies in rodents (generally rats) and non-rodents (generally rabbits). This study serves to determine potential adverse effects of the test article on the pregnant female animal and on development of the embryo and fetus. Maternal and fetal exposure are also estimated by toxicokinetic analyses. The pregnant females are administered the test article from implantation of the embryo to the closure of the hard palate of the fetuses (rat: gestation days 6 to 17; rabbit: gestation days 7 to 20)
- Segment III: Peri- and postnatal study generally in rodents (mainly rats). This study serves to determine potential adverse effects of the test article on the pregnant and lactating females and on the functional and morphological development at the offspring. Only FO females (= adult females at the beginning of the study) are administered the test compound from implantation of the embryo to the end of lactation
The presentation on Toxicologic pathology of the male reproductive system - Basis shows that this system is rather complex regarding structural elements and regulation. Often more than one male reproductive target is adversely affected by diverse chemicals and drugs. It is important to understand class effects and to know that endocrine adverse effects are often species-specific and with little relevance to man

The presentation on Toxicologic pathology of the male reproductive system – Practice underlines that the male reproductive system is key for survival of a species and that the declining number and quality of human sperm are a concern. The latter is most likely caused by unhealthy diet, sedentary lifestyle, exposure to various man-made chemicals including pesticides or by smoking, stress, etc. Thus, while the number of sperm in e.g. rats can decrease by 90 %, before successful insemination is affected, that of human males is generally just above thresholds. This means that a Segment I study is relatively insensitive, which can be compensated by large overdosing. However, post-mortem examinations including organ weights and histopathology as conducted in regular toxicity studies are relatively sensitive for detecting male reproductive toxicity. This can be further enhanced by the analysis of sperm numbers

In conclusion: preclinical potentially adverse effects on the male reproductive system are not uncommon and of concern. They must undergo a sound and comprehensive scientific assessment e.g. regarding safety ratios, reversibility (survival of spermatogonia) and monitorability in man. Standard PCS studies are often sufficient, but good tissue fixation and staining particularly of the testis and expert histopathological examination including testicular staging are important. Immature test animals are confounding. The established mode of action (MoA) including identification of the primary target is important. This may need early time points, time-course studies and hormonal measurements. The ultimate proof that preclinical effects on male reproduction are without consequences for man are possible often only in man with early markers

For details see also specific guidelines, e.g. ICH S5(R3)

Genotoxicity testing. There are two options according to ICH S2(R1):

Option 1

Option 2

Bacterial gene mutation (generally no repeat needed)

Maximum dose level 5000 μg/plate (or 5 μL/plate for liquid test substance), if not limited by solubility or cytotoxicity

One in vitro mammalian cell test:

Chromosome aberrations or
Mouse lymphoma assay or
Micronucleus assay (MLA)

At 1 mM top concentration or 0.5 mg/mL or at most 50 (for MLA 80-90) % cytotoxicity, if not limited by solubility

No in vitro assay in mammalian cells

One in vivo micronucleus test

Preferably integrated into rodent toxicity study

Two in vivo tests, e.g.

In vivo MLA in hematopoietic cells plus
2^nd in vivo endpoint and/or tissue

Preferably integrated into rodent toxicity study

Note that a compound might have a carcinogenic potential in the absence of detectable genotoxicity

See also study designs for additional details.