© 2018 Envigo envigo.com

Mandy Horn Manager, Veterinary Science, Research and SupportMark Blee Director, Toxicology

From nose to tail: non-clinical safety assessment success factors in rats

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We are a global contract research products and services company dedicated to helping our customers achieve the potential of their products which improve human life, advance animal welfare, and protect the environment and global food security.

Who we are

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+ Introduction + Animal models and their differences+ Diet considerations+ A case study – transitioning 2-year studies to a different rat

strain and diet – lessons learned+ Summary

Overview

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Introduction

+ Preclinical toxicity studies necessary to attain regulatory approval of new drugs and chemicals

+ Extensive safety analyses are completed which may include 2-year studies

+ Animal model selection is key to drug development success

+ Preclinical studies are one part of the drug development timeline

Introduction

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Introduction

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Graphic Courtesy of the American Association of Cancer Research 2011 Cancer Progress Report

+ Most toxicology studies must be conducted in one rodent and one non-rodent species + Study types include Pharmacokinetic (PK), Maximum Tolerated Dose (MTD), Dose

Range Finding (DRF), 28-d, as well as sub-chronic and chronic at 3, 6, 9, or 24 months

+ Reprotoxicity studies+ Purpose of this presentation will focus on chronic studies

Preclinical study types

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Animal models and their differences

+ Genetic similarity (>90% of genome)

+ Increased sample volumes compared to mice (blood and tissues)

+ Robust historical control data

+ Consider ability to extrapolate study data to human risk relevance or irrelevance

What makes a good rodent model?

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+ Improved animal welfare+ Number of animals needed to complete the study

+ Economic benefit+ Less compound use+ Fewer animals+ Reduced labor

+ Housing efficiency

+ Reduced research variables+ Historical control data available+ Robustness of data due to sample size

Benefits of a good animal model

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+ Several commercially available rat models; well characterized with supporting published literature available

+ F344+ Inbred, utilized for several years by NTP as preferred model (until 2006)+ Concerns over high incidence of select tumors

+ Sprague Dawley+ Outbred, well-characterized+ Good reproductive characteristics; widely used for reprotoxicity studies+ Historically utilized as model of choice in US, large HCD exists

+ Wistar Han+ Outbred, well-characterized+ Robust survival, smaller body size, and lower overall tumor burden

Select animal models available

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104-wk growth curve comparison

0

100

200

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500

600

700

800

900

7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 100

103

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Weeks of AgeRccHan:WIST Males CD Males RccHan:WIST Females CD Females

Bod

y W

eigh

t (g)

800 g

594 g

510 g

385 g

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104-wk survival curve comparison

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 100

103

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Perc

ent S

urvi

val

Weeks of Age

RccHan:WIST Males CD Males RccHan:WIST Females CD Females

73%

67%

38%

31%

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Tumor burden comparison

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Weber, K, et al. (2011)

Weber, K. (2017)

Cumulative tumor incidence comparison

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0

10

20

30

40

50

60

70

80

90

100

Thyroid Testis Pituitary Mammary Lymphoma Liver Adrenal

(%) I

ncid

ence

RccHan:WIST Male RccHan:WIST Female CRL:CD Male CRL:CD Female

*RccHan®:WIST data – internal HCD* Crl:CD data-www.crj.co.jp

+ Chronic Progressive Nephropathy*+ Decreased incidence with restrictive caloric intake

+ Vasculitis* (+/- vascular mineralization)+ Aorta and tongue are predilection sites – with arterial rupture and/or dysphagia and

refusal of food intake possible sequelae+ Progressive cardiomyopathy (PCM)*

+ Variable expression of background or incidental disease, like PCM in SD rats is a fairly common observation in toxicologic pathology, and modest group size in standard study designs is commonly used to manage these variable effects….This particularly high incidence, together with other factors, prompted GSK to change the rat strain used in routine toxicity study from the Sprague-Dawley to the Han Wistar. (Chanut et al., 2013)

+ Corneal dystrophy & mineralization+ Especially important if eye is target

+ Reproductive lesions+ Interstitial/Leydig Cell hyperplasia (highest in F344 rats)+ Uterine Squamous Cell Metaplasia (highest in SD rats)

Selected key and common nonproliferative/nonneoplasticlesions with lower incidence in RccHan®:WIST rats

*Common cause of death in severe cases

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+ F344 and SD rats have higher tumor incidences than RccHan:WIST+ RccHan:WIST studies have lower incidences of tumor-bearing animals

+ Mammary Gland Tumors* (especially fibroadenomas)+ Pituitary Pars Distalis Adenomas*+ Adrenal Gland Tumors*

+ Pheochromocytomas (medulla) + Adenomas (cortex)

+ Higher incidence of thyroid gland follicular tumors, thymomas, and lymph node vascular neoplasms in RccHan:WIST rats (vs. SD rats), but these are generally focal neoplasms, non-contributory to death

Selected key and common proliferative/neoplastic lesions with lower incidence in RccHan®:WIST rats

*Frequent cause of death

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+ Reprotoxicology+ High fecundity (11-14 pups/litter)+ Low variation in reproductive success between generations+ ~50%:50% gender distribution in litters+ Rare congenital abnormalities

+ Neurotoxicology+ SD rats more susceptible to MAM-induced learning and

memory impairments and neurological damage (Zmarowski et al., 2012)

+ Inhalation+ SD rats are larger (higher body weights) and have higher

incidence of palpable masses (especially mammary tumors), therefore if inhalation tubes are used, confinement becomes difficult in longer term studies

Specialized studies

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+ Reduction in compound usage by nearly 30%+ Due to lower body weight and improved survival to two years

+ Fewer animals necessary at study start due to improved survival, allowing for decreased per diem and overall housing and husbandry costs

+ Labor reduction due to ability to pair house and feed ad libitum throughout the study

Economic benefits

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Diet considerations

+ Natural ingredient+ Agricultural commodities

+ Corn, wheat+ Soybean meal, corn gluten

meal, fish meal+ Relatively unrefined+ Chemically complex

+ Refined+ Further processed

+ Corn starch, sucrose, cellulose+ Casein + Vegetable oil, animal fats

+ Food grade+ Chemically simple

Diet classification

Ingredient composition

+ Standard+ Colony management – breeding,

maintenance, aging+ Research non-specific

+ Custom+ Nutrient control+ Induce disease+ Dose animal

+ Medicated+ Veterinary purpose

Use/purpose

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+ Nutrients+ Protein+ Energy+ Improper/imbalanced micronutrients

+ Contaminants+ Heavy metals+ Mycotoxins+ Pesticides+ Nitrosamines

+ Non-nutrients+ Phytoestrogens

Diet as source of variation in toxicology studies

Component

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Components of fixed formulation process

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Fixed formula diets contain the same ingredient, in the exact same quantities, in every batch of diet

Quality practices – ingredient sourcing through manufacturing

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+ Ingredients from approved regional suppliers + Each and every load is sampled and tested upon arrival+ All specifications, including NIRS & mycotoxin screening,

must be met before product is accepted and the truck is allowed to unload

+ Followed by automated diet production; computer controlled mixing with accurate accounting of ingredient usage

+ Quality systems are ISO 9001:2015 certifiedTrust but verify

Expectations for nutrient variability (CV, %) – standard diets

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Protein, %+ Expected Variation

+ Protein – <5%+ Fat – <10%+ Minerals – 5-15%+ Vitamins – 5-25%

+ QA – protein, fat, crude fiber, ash, calcium & phosphorus

+ Can be measured 02468

101214161820

201420162018

Dietary protein

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Diets in common use supply protein well in excess of requirements

+ Reproduction – 18% protein+ Growth – limited benefit past 13% protein+ Maintenance - ~7% protein

Excessive protein/energy associated with renal damage+ Appear early + Decreases survival + Depends on strain+ May lead to questionable toxicology data

Effects of dietary protein on kidney function

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30

40

50

60

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1 52 103Weeks on Study

Wat

er c

onsu

mpt

ion,

gm

/rat/d

ay

Water consumption pattern of male Wistar Rats fed 23% protein diet

1 2 3 423% Protein Diet 0 48 42 1014% Protein Diet 21 70 9 0

0

20

40

60

80

Inci

denc

e, %

Severity of Nephropathy(male F344 rats fed ad lib for 2 years)

Rao, 2002, Toxicological Pathology 30:651-656

Effect of varying degrees of diet restriction on survival of Sprague Dawley rats

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0

10

20

30

40

50

60

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90

25 50 75 100

Energy Intake (Kcal/d)

106-

wee

k Su

rviv

al (%

)

Ad Libitum intake

80% DR

75% DR

50% DR

Males

Females

Keenan et al., 1999 Toxicological Sciences 52(Suppl) 24-34

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60

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11 15 19 23 27 31 35 39 43 47 51 55 59 63 67 71 75 79 83 87 91 95 99 103

Bod

y W

eigh

t, g

Age, week

Body Wt Survival

Male Hsd:Sprague Dawley®SD® growth and survival curves

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Surv

ival

, %

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90

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50

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30

20

10

0

BW data is mean ± 2SD N = 200 Diet = Teklad 2014S starting at 8 weeks of age

+ Body weight plateaus at ~550 g without diet restriction; compare to CD® IGS rat which are 100-200 g heavier when fed more typical standard diet

+ Survival at 2 years ~68%; compare to typical 2 year survival in CD® IGS rat of ~35-40%

+ Mycotoxins+ Aflatoxins, vomitoxin, fumonisin

+ Nitrosamines

+ Heavy metals+ Lead, Mercury, Arsenic, Cadmium

+ Pesticides

Some contaminants of importance in laboratory diets

Type+ Food refusal; chronic

consumption leads to organ damage, cancer

+ Chronic consumption –tumors; modern day fish meal has lower levels

+ Chronic consumption –organ damage

+ Can act as endocrine active compounds

Significance

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Phytoestrogens – recommendations

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OECD Guideline For the Testing of Chemicals (2009)451: Carcinogenicity Studies, Adopted 7 September 2009452: Chronic Toxicity Studies, Adopted 7 September 2009453: Combined Chronic Toxicity/Carcinogenicity Studies (draft)

Housing and feeding conditionsThe diet should meet all the nutritional requirements of the species tested and the content of dietary contaminants including but not limited to pesticide residues, persistent organic pollutants, phytoestrogens, heavy metals, and mycotoxins, that might influence the outcome of the test, should be as low as possible.

Guide for the care and use of Laboratory Animals, 8th ed.

Contaminants such as pesticide residues, heavy metals, toxins, carcinogens, and phytoestrogens may be at levels that induce few or no health sequelae yet may have subtle effects on experimental results (Thigpen et al. 2004) Page 65.

Phytoestrogens – why should researchers be aware?

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+ ER widely distributed in tissues

+ Even with lower binding affinity, isoflavones have considerable access to receptors by virtue of high serum levels

+ Many areas of research affected

http://endocomprehensive.blogspot.com/2013/11/estrogen-receptors-importance-in.html

estradiol

genistein

daidzein

+ Primarily bind ERβ+ SERM - can act as estrogen

agonist or antagonist+ Non-estrogenic mechanisms

Isoflavone levels in soybean meal and diets containing low, medium and high levels of soybean meal

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+ Isoflavone range in rodent diets containing soybean meal ~100-700 mg/kg+ 2-fold variation in “same” diet over time

N = 51 over 13 years

Soybean meal

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Low Medium High

Gen

iste

in +

dai

dzei

n (m

g/kg

)

Diets

Estrogenicity and anti-estrogenicity of dietary soybean meal in mouse uterus bioassay

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Dietary Group

(d 16-23)n Relative uterine weight

(mg/kg bw) Soy Effect

SOY + 32 0.87 ± 0.04 + 0.11

SOY - 33 0.76 ± 0.03

SOY+ with DES (6ug/kg) 18 1.01 ± 0.12 -0.48

SOY – with DES (6ug/kg) 22 1.49 ± 0.11

Mäkelä et al. 1995 J Nutr 125:437-445

+ In chemically-induced models, the risk of tumors (incidence, latency,tumor number) was substantially decreased in the presence of genistein (Barnes, 1995)

+ Significance for regulatory carcinogenicity studies, as the absence of phytoestrogens should be beneficial for “normal” development of tumors in the control and dosed animals, within the time period of the study

+ Essential that the diet promotes healthy longevity, without masking any treatment/compound effects

Phytoestrogens and carcinogenicity studies

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+ Literature – no simple threshold for physiological effects+ Hard to predict the magnitude or direction of response+ Reduces translatability of model

Take home - Phytoestrogens

+ Nutrients+ Protein+ Energy+ Improper/imbalanced micronutrients

+ Contaminants+ Heavy metals+ Mycotoxins+ Pesticides+ Nitrosamines

+ Non-nutrients+ Phytoestrogens

Diet as source of variation in toxicology studies

Component+ Proper selection

+ For life stage and study goals+ Uniformity+ Fixed Formulation

+ Ingredients+ Selection+ Screening+ Reports available+ Fixed formulation

+ Ingredients+ Selection+ Minimize substances with known

biological activity+ Fixed Formulation

Solution

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A case study – transitioning 2-year studies to a different rat strain and diet –lessons learned

Strain Route Diet Live Completion 2007-2017

Sprague –Dawley (CR SD IGS

Diet RM1 4 (4)

Teklad

Oral (gavage) RM1 12 (15)

Teklad 1 (1)

Inhalation RM1 5 (7)

Teklad

Other RM1 2 (3)

Iv,sc, vg Teklad 2 (2)

Background control data no. of studies since 2007

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Background control data – no. of studies since 2007

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Strain Route Diet Live Completion 2007-2017

RccHan®:WIST (Envigo)

Diet RM1 1 (1) 5 (5)

Teklad 1 (1)

Oral (gavage) RM1 11 (14)

Teklad 4 (8)

+Total number of rat carcinogenicity studies performed in the UK since 2007 (number of control groups):

+Sprague Dawley: 26 (32)+Han Wistar: 22 (29)

Total number of carcinogenicity studies since 2007

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Rat Strain

Housing

Gang Single All studies

M F M F M F

Sprague-Dawley

MeanRange

n

4628-71

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3822-57

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4340-48

3

2718-33

3

4628-71

52

3718-57

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RccHan®:WIST

MeanRange

n

7360-84

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6550-82

40

7269-75

2

6463-65

2

7360-84

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6550-82

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Percentage survival at termination of carcinogenicity studies since 1996 – all routes

A few studies with Sprague-Dawley rats were terminated before reaching 104 weeks. This requires a discussion with the regulatory authorities.

In an attempt to reach Week 104, the group size for Sprague-Dawley studies is usually 65M & 65F, whereas Han Wistar rat studies are at 52M & 52F.

Survival issues

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Gang housing with fun tunnel

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+ Animal model selection key to study success and has economic benefits as well+ Consider survival and body weight throughout the study+ Interpretation of study data including non-proliferative

and proliferative rare and common findings

+ Nutrient and non-nutrient components impact study success and should be considered critical to study development

Summary

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