Cesare Maltoni Cancer Research Center Annual Ramazzini Days Sprague Dawley rats from the CMCRC/RI: a suitable model for studying different endpoints in experimental toxicology Eva Tibaldi Cesare Maltoni Cancer Research Center Good morning to everybody, thank you for inviting me. Today I'm going to talk about the Sprague Dawley rats from the CMCRC: a suitable model for studying different endpoints in experimental toxicology Carpi, October 28th, 2016

IN CARCINOGENICITY BIOASSAYS 40 YEARS OF EXPERIENCE IN CARCINOGENICITY BIOASSAYS The CRC has more than 40 years of experience in using the animal model to conduct long term bioassays. All the experiments are conducted following an highly standardized protocol in accordance with the good laboratory practices. 2 2

Consistency of the animal model and experimental conditions Animal model: family tree of SD rats from the 1970’…..until now (more than 200.000) Housing conditions (cages, conventional room) Diet (periodically analyzed for nutritional components and determination of contaminants) Start and duration of the experiment (for carcinogenicity at least 130 weeks or more) Conduct of the biophase (measurements of water and food consumption, body weight, behaviour and clinical control Conduct of the histopathology phase (complete necropsy, fixation, embedding, cutting and staining) Microscopic examination (harmonization of the diagnoses with international classification criteria) Final report (biophase and histopathology tables, statistics) A prerequisite of our laboratory is the consistency over the time both of our animal model and the experimental conditions. First of all the animal used is the SD rat; we have an internal breeding at the CRC and the colony originates in the 70's when Professor Maltoni started the first experiments on Vinyl Chloride and the animals were purchased the Charles River company. The advantages of this strain are the calmness and ease of handling; the excellent reproductive performance and the long life (mean 3 years) and for these reasons this model is used in all disciplines of biomedical research (including carcinogenicity, toxicology, reproduction, pharmacology and behavioural studies). Same housing conditions were maintained: for example the number of animals in cages is 3, the conventional room are maintained at constant temperature, relative humidity and light-dark cycle. The animals were fed with pellets "type Corticella" prepared with the same formula in last 40 years and watered with tap water. The diet and water are periodically analyzed for nutritional components and determination of contaminants of various origin such as pesticides, heavy metals, phytoestrogens, micro-organisms. Peculiar characteristics are the start and the duration of the experiment (the start could be from prenatal life, at 6-8 weeks of age or in adult) and for the carcinogenicity studies the end of experiment is at least 130 weeks or more (to reach the full expression of carcinogenic potential of the agent studied). The same standardized procedures are followed in the in vivo and histopathology phase. For example we perform periodic measurements of water and food consumption and body weight, behavioural and clinical control to check the state of health and detect macroscopic lesions. Then we perform a complete necropsy and all tissues are fixed, embedded in paraffin blocks and from blocks we obtain sections stained with hematoxilin-eosin for microscopical examinations. All slides are screened by a first pathologist and peer reviewed by same supervisor senior pathologist and all diagnoses are harmonized following international classification criteria. Finally a final report including biophase and pathology tables with statistical analyses was prepared. 3 3

BODY WEIGHT and SURVIVAL In order to prove the consistency of our animal model over the time we investigated the body weight and survival of groups of untreated animals, that represent the historical control of our strain of rats. 4 4

HISTORICAL CONTROL: data at 104 weeks control groups of 158 experiments from 1969 to 2011 N. of animals Mean body weight Mean survival Males 7,854 533.2 g 45.7 % Females 8,202 376.4 g 51.1 % Total (M+F) 16,056 47.8 % We analyse the control groups of 158 experiments from 1969 to 2011 considering more than 16 thousand of animals. To compare our data with other laboratories the body weight and survival are considered at 104 weeks that corresponds to the end of experiments for carcinogenicity studies.

MEAN BODY WEIGHT (g) IN MALE AND FEMALE RATS As you can see in this graphs the trend of body weights in both M and F is very constant over the years, for M the mean body weight is between 500 and 600 g while for F is between 350 and 400 g.

MEAN SURVIVAL (%) IN MALE AND FEMALE RATS The trend for the survival is quite similar in both M and F and ranges between 40 and 50%. In general the survival in F is higher than in M.

Comparison: Body weight and survival at 104 weeks Now I would show you the comparison between our results and data from other laboratories. In this slide we observe that the body weight values are very similar comparing different facilities with a little difference for the Charles River Japan strain. as regarding the survival, the incidences are quite similar. For female Fischer rat there is an higher incidence compared to other animals. I want to highlight you that at 104 weeks the 40-50% of animals are still alive. (So ending up the experiments at 104 weeks, we could lose important data about the potential carcinogen of the studied agent). (I) (II) NTP data: Toxicol Pathol. 2003 Nov-Dec;31(6):674-81. Haseman JK et al. Effect of diet and animal care/housing protocols on body weight, survival, tumor incidences, and nephropathy severity of F344 rats in chronic studies. Charles River data: (I) Hum Exp Toxicol. 1993 Mar;12(2):87-98. Nohynek GJ, et al. Fat, frail and dying young: survival, body weight and pathology of the Charles River Sprague-Dawley-derived rat prior to and since the introduction of the VAFR variant in 1988 France laboratory; (II): Report on several studies dated 2004;Charles River Japan: Report on biological reference data on CD SD IGS rats 2001, Yokohama; Harlan: Fundamental and Applied Toxicol 1996; 33, 196-211, Pettersen JC et al. A 2-Year Comparison Study of Crl:CD BR and Hsd:Sprague-Dawley SD Rats

INCIDENCE AND TYPE OF TUMORS 1984-1994 versus 1995-2004 My next point is: how is changed the incidence of the tumors in our colony over the time? We compared the data of two decades 1984 -1994 versus 1995 -2004. These periods are referred to the start of biophase (the elaboration of the pathology data for the following decades is still ongoing). 9 9

ANIMAL BEARING BENIGN TUMOR IN MALE AND FEMALE Males Females 1984-1994 1995-2004 1984-1994 1995-2004 As you can see the distribution by age and the incidences of animals bearing benign tumor are similar in the two decades for both males and females. Male (%) 60.75 58.13 Female (%) 76.74 75.49 1984-1994 N. of animal: 2265 ; 1995-2004 N. of animal: 1428

INCIDENCE OF BENIGN TUMOR IN MALE AND FEMALE RATS (%) 1984-1994 1995-2004 M F M F Fibroma and Fa of the mammary gland Adenoma of the pituitary gland Pheocromocytoma of the adrenal gland Polyp of the uterus Islet cell adenoma of the pancreas Interstitial cell adenoma of the testis 4.24 44.50 24.33 41.91 33.86 24.98 - 15.57 8.43 3.96 7.28 - 4.47 49.37 28.60 30.25 20.30 21.36 - 21.50 8.94 3.43 3.90 - In this slide are reported the most frequent benign tumors observed in our colony. They are Fibroma and Fa of the mammary gland, Adenoma of the pituitary gland, Pheocromocytoma of the adrenal gland, Islet cell adenoma of the pancreas, Polyp of the uterus and Interstitial cell adenoma of the testis.

INCIDENCE (%) OF MAIN BENIGN TUMORS IN MALES The incidences for the various types of benign tumors are quite similar comparing the two decades. We observe a marked decrease in Pheocromocytoma, a little decrease in Interstitial cell adenoma and a little increase of Pituitary gland adenoma in males.

INCIDENCE (%) OF MAIN BENIGN TUMORS IN FEMALES While for F there is a marked decrease of Pituitary gland adenoma and a little increase in Mammary fibroadenomas and polyp of the uterus.

ANIMAL BEARING MALIGNANT TUMOR IN MALE AND FEMALE 1984-1994 Males Females 1995-2004 1984-1994 1995-2004 Also for animals bearing malignant tumors the distribution by age and incidences are similar in the two decades. Male (%) 41.24 38.33 Female (%) 40.06 44.89 1984-1994 N. of animal: 2265 ; 1995-2004 N. of animal: 1428

INCIDENCE OF MALIGNANT TUMOR IN MALE AND FEMALE RATS (%) 1984-1994 1995-2004 M F M F Haemolymphoreticular neoplasia Carcinoma of the mammary gland Carcinoma of the ear ducts Osteosarcoma of the head Malignant tumor of the uterus 20.44 13.10 0.49 9.94 5.21 6.02 4.86 2.90 - 6.47 18.95 14.36 0.35 9.73 3.19 6.09 6.60 4.97 - 6.93 In this slide are reported the most frequent malignant tumors observed in our colony. They are Haemolymphoreticular neoplasia, Carcinoma of the mammary gland, Carcinoma of the ear ducts, Osteosarcoma of the head and Malignant tumor of the uterus.

INCIDENCE (%) OF MAIN MALIGNANT TUMORS IN MALES The incidences of malignant tumors in both Males and Females are stationary for the two decades. In males we observe a little increase in osteosarcomas and a decrease in ear duct carcinoma.

INCIDENCE (%) OF MAIN MALIGNANT TUMORS IN FEMALES In females we observe a little increase in osteosarcomas and HLRN.

COMPARISON: INCIDENCE (%) OF MAIN BENIGN TUMORS IN MALES At this point we made a comparison of our tumor incidences with data reported in literature regarding studies conducted by other laboratories using Fischer rats or SD rats from Charles River. For male benign tumor the Fischer rat presents a very high incidence of Interstitial cell adenoma of the testis with respect to SD rats. This excess of tumors is one of the reason why 10 years ago the NTP changed Fischer rat strain with the SD. The Ch River SD rat presents a higher incidence of pituitary gland adenoma with respect to our strain of SD rat. For NTP F344 data: Haseman et al 1985, “Neoplasms observed in untreated and corn oil gavage control groups of F344/N rats and B6C3F1 mice”, JNCI and data from the book Pathology of the Fischer Rat: Reference and Atlas For CRC SD data: data on historical control from experiments conducted in 1984-1994 For CH RIV SD data: Mc Martin et al 1992, “Neoplasms and related proliferative lesions in control Sprague-Dawley Rats from Carcinogenicity studies. Historical data and diagnostic considerations” Toxicol Pathol and from Charles River “Compilation of Spontaneous Neoplastic Lesions and Survival in Crl:CD (SD) Rats from Control Groups” March 2004

COMPARISON: INCIDENCE (%) OF MAIN BENIGN TUMORS IN FEMALES For female benign tumor a higher incidence of pituitary adenoma and a lower incidence of polyp of the uterus are observed in Ch River SD rats, while in our strain there is a higher incidence of pheocromocytomas of the adrenal gland with respect to other animals. For NTP F344 data: Haseman et al 1985, “Neoplasms observed in untreated and corn oil gavage control groups of F344/N rats and B6C3F1 mice”, JNCI and data from the book Pathology of the Fischer Rat: Reference and Atlas For CRC SD data: data on historical control from experiments conducted in 1984-1994 For CH RIV SD data: Mc Martin et al 1992, “Neoplasms and related proliferative lesions in control Sprague-Dawley Rats from Carcinogenicity studies. Historical data and diagnostic considerations” Toxicol Pathol and from Charles River “Compilation of Spontaneous Neoplastic Lesions and Survival in Crl:CD (SD) Rats from Control Groups” March 2004

COMPARISON: INCIDENCE (%) OF MAIN MALIGNANT TUMORS IN MALES For what concern the incidences of malignant tumors there is some variability among the strains in both M and F. In the CRC SD rat there are more Osteosarcomas and ear ducts carcinomas compared to other animals, while the Fischer rat shows an high incidence of HLRN and the Ch Riv SD rat has a low incidence of HLRN. . For NTP F344 data: Haseman et al 1985, “Neoplasms observed in untreated and corn oil gavage control groups of F344/N rats and B6C3F1 mice”, JNCI and data from the book Pathology of the Fischer Rat: Reference and Atlas For CRC SD data: data on historical control from experiments conducted in 1984-1994 For CH RIV SD data: Mc Martin et al 1992, “Neoplasms and related proliferative lesions in control Sprague-Dawley Rats from Carcinogenicity studies. Historical data and diagnostic considerations” Toxicol Pathol and from Charles River “Compilation of Spontaneous Neoplastic Lesions and Survival in Crl:CD (SD) Rats from Control Groups” March 2004

COMPARISON: INCIDENCE (%) OF MAIN MALIGNANT TUMORS IN FEMALES In females we observe the same trend for Osteosarcomas and ear ducts carcinomas in CRC SD rat and for HLRN in Fischer and Ch Riv SD rat, while the Ch Riv SD rat has an high incidence of malignant mammary carcinoma. For NTP F344 data: Haseman et al 1985, “Neoplasms observed in untreated and corn oil gavage control groups of F344/N rats and B6C3F1 mice”, JNCI and data from the book Pathology of the Fischer Rat: Reference and Atlas For CRC SD data: data on historical control from experiments conducted in 1984-1994 For CH RIV SD data: Mc Martin et al 1992, “Neoplasms and related proliferative lesions in control Sprague-Dawley Rats from Carcinogenicity studies. Historical data and diagnostic considerations” Toxicol Pathol and from Charles River “Compilation of Spontaneous Neoplastic Lesions and Survival in Crl:CD (SD) Rats from Control Groups” March 2004

A HUMAN-EQUIVALENT MODEL 15 years ago, when Cesare Maltoni passed away, a conference in his honor was organized at the NY academy of sciences and in this occasion his co-workers presented data on tumor incidence of our historical control SD rats compared to data on human population, in order to prove if our strain could represent a good human-equivalent animal model. 22 22

HUMAN-EQUIVALENT MODEL: CUMULATIVE PREVALENCE BY AGE AT DEATH 1,114 people with malignant tumors (out of 2,560 autopsied men and women deceased at the Hospital of Trieste, in 1989) 1,212 Sprague-Dawley rats with malignant tumor (out of 3,051 necropsied male and female untreated rats (from control groups) More than one thousand people with malignant tumor deceased at the Hospital of Trieste in 1989 are compared to the same number of SD rats bearing malignant tumor from control groups. The results show that the cumulative prevalence of malignant tumor by age at death is very similar between humans and rats. It is noteworthy the fact that the most part of tumors are observed after 65 years of age in humans corresponding to 104 weeks of age in rats. (So ending up the experiments at 104 weeks, we could lose important data about the potential carcinogen of the studied agent). (The results of several studies conducted by IR have shown an increased incidence of various cancers occurred after 104 weeks of age; for example the thyroid tumors in the pesticide Mancozeb or the hamangiosarcoma of the liver in Vynil chloride) 80% of tumors occur in humans after 65 years, corresponding to 104 weeks of age in rats 23

CONCLUSIONS The RI colony of SD rats has consistent biological characteristics over time. Our animal model represents a "human-equivalent" model in terms of incidence, natural history and age distribution of various types of cancer. Our strain has a low background of spontaneous hormonally-induced tumor incidence (ovary, thyroid, testis, mammary gland). Therefore this model is able to provide important information on non-neoplastic and neoplastic waiting and is suitable for studying different types of endpoints in experimental toxicology. At the end of my presentation we can conclude that our colony has consistent biological characteristics over the time and our animal model represents a good "human-equivalent" model as to incidence, natural history and age distribution of various types of cancer. Finally our strain has a low background of spontaneous hormonally-induced tumor incidence (for ovary, thyroid, testis, mammary gland). so therefore this model is able to provide important information on non-neoplastic and neoplastic waiting and is suitable for studying different types of endpoints in experimental toxicology. (for example the model can be used for studies on endocrine disrupting agents).

“The reward of great men is that, long after they have died, one is not quite sure that they are dead” Jules Renard , 1864 -1910 Thank you for the attention. I'd like to thank all my colleagues for the work we have done together over the years. Any questions? 25