Carcinogenicity Evaluation: Comparison of Tumor Data from Dual Control Groups in the Sprague–Dawley Rat

Introduction

Preclinical drug testing has benefited from a range of national, region-specific (e.g., Europe, United States, and Japan) or international directives, regulations, or guidelines. Furthermore, the emergence of the International Conference on Harmonization (ICH) has resulted in guidance documents that have helped to eliminate region-specific differences. However, until recently, a continued difference has been in the use of double control groups in Europe for carcinogenicity studies vs. the common use of only single control groups in other regions, such as the United States.

A review of guidance documents relating to carcinogenicity studies highlights why this situation occurred. Until recently, European guidelines indicated 2 control groups dosed with vehicle (Guidelines Volume 3B, 1998). However in the United States, 1 concurrent control group is mentioned (FDA Red Book, 1982) that agrees with OECD requirements (OECD, 1993); in Japan, a negative control group should be included (Japan, 1999). Relevant ICH documents do not mention the number of required control groups (ICH S1C, 1995; ICH S1A, 1996; ICH S1C(R), 1997; ICH S1B, 1998). As a consequence of these differences, some pharmaceutical companies have routinely included dual control groups to support global introduction of a new drug. It appears that single control groups may now become more popular with the recent clarification in Europe that 1 control group of the same number for each sex dosed with the vehicle by the same route is acceptable (CPMP/SWP/2879/00). Interestingly, a recent review of practices within carcinogenicity studies in the United States has indicated that 9/15 companies that responded to a questionnaire use 2 vehicle control groups (Perry and Menton, 2004).

Carcinogenicity studies obviously have an important role in identifying tumorigenic potential in animals, as part of the assessment of risk in humans for new drugs. However, little published data are available on how the use of 2 control groups has assisted this process. It has been pointed out that additional controls give a better assessment of how rare each tumor type is and allows a more accurate comparison to historical control data (Fairweather et al., 1998). Additionally, it is said that the results from 2 identical controls can be used as a mechanism for identifying the extent of control variability and to help evaluate the biological significance of increases in tumor incidence in the treated groups (CDER, 2001).

This paper presents data from 13 in-house carcinogenicity studies using Sprague–Dawley rats with 2 identical control groups. All the studies involved oral administration and were performed in the period 1991–2002. As well as examining potential tumor incidence differences between the dual controls, the paper also provides an update on the range and extent of background neoplasms in this commonly used rat strain and includes comment any on genetic drift.

Materials and Methods

Selection of studies for evaluation from our in-house database was limited to carcinogenicity investigations in gang-housed Sprague–Dawley (Crl: CDBR strain) rats with 2 identical control groups using oral administration (gavage tube or dietary) in the period 1991–2002. A total of 13 studies were identified giving a total of 765 males and 765 females for each control group. Group sizes ranged from 50–65 animals/sex. Treatment comprised gavage dosing at 5 or 10 mg/kg of vehicle material or dietary intake of standard chow. Rats were treated for a 2-year period (actual period 98–105 weeks).

Rats of approximately 6 weeks of age at the start of dosing were obtained from Charles River UK and were housed 5 to a cage. They were fed SQC Rat and Mouse Maintenance Diet No. 1 (Special Diets Service Ltd., United Kingdom) ad libitium and had free access to water. Animals were maintained on a 12-hour light/dark cycle at 19–25°C and 40–70% relative humidity. At the end of the treatment period, complete necropsies were conducted on all the control animals and tissues were fixed and embedded in paraffin wax, sectioned (nominally 5 μm) and stained with hematoxylin and eosin. Tissues routinely examined in the 13 studies were adrenals, brain, cecum, colon, duodenum, eyes, femur with bone marrow and articular surface, gross lesions, Harderian glands, heart, ileum, jejunum, kidneys, liver, lungs with mainstem bronchi, mammary gland (female), mandibular and mesenteric lymph nodes, muscle, esophagus, optic nerves, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerves, seminal vesicles, skin, spinal cord (at 3 levels), spleen, sternum with bone marrow, stomach, testes + epididymides, thymus, thyroids + parathyroids, tissue masses, tongue, trachea, urinary bladder, uterus, and vagina.

Survival after 2 years of treatment was measured. Rats were examined for nonneoplastic and neoplastic findings. Tumor identification was based on in-house nomenclature, which followed that commonly used elsewhere, e.g., International Agency for Research on Cancer (IARC) and Society of Toxicologic Pathologists (STP). To allow for in-house consistency, “older” synonyms for some tumors have been used for some neoplasms. Statistical evaluation was performed for differences between the 2 control groups (designated control I and control II). Survival and tumor incidence were compared using a 2-sided chi-squared test. If the incidences were shown to be not significantly different, then the 2 groups were combined for comparison with drug-treated groups in each of the presented carcinogenicity studies. All studies were performed to Good Laboratory Practice (GLP).

Results

Survival at study end is given in Table 1. Overall, mean survival was similar between the 2 groups with 38–57% and 32–60% survival seen for males and 22–48% and 25–57% survival seen for females in control groups I and II, respectively. Generally, only small differences between the 2 control groups were seen for individual studies with no statistically significant differences in survival. When survival was broken down to 4-year periods of 1991–1994, 1995–1998, and 1999–2002, there was no obvious time effect on survival values. Across all studies, the main causes of death were consistent with those generally associated with aging laboratory rats of this strain and comprised pituitary and skin/appendage tumors in both sexes (with the latter predominantly in males) plus mammary tumors predominantly in females.

Common nonneoplastic lesions in decedents and rats surviving to termination are shown in Table 2. Similar findings were seen for both control groups across all the studies with no obvious group differences seen. The findings were consistent with the spectrum associated with aging rats of this strain and included inflammatory (leukocyte) cell foci in several organs, chronic nephropathy in the kidney, vacuolation in the liver, C-cell hyperplasia in the thyroid, myocarditis/fibrosis in the heart, and foamy histiocytes in the lung.

A summary of tumor incidences is given in Table 3 with details of major organ or common and less common tumors shown in Tables 4 and 5, respectively. Notable interstudy differences in tumor incidences between dual control groups are presented in Table 6. Table 3 shows that the total number of neoplasms was similar for male and female control groups with a slightly higher tumor burden in females compared to males. Across all the groups, the malignant tumor incidence was 12–15%. When tumor incidences were broken down to 4-year periods of 1991–1994, 1995–1998, and 1999–2002, results were generally similar although a slight trend for a decreased incidence in tumor burden was seen for males vs. a slight increase in burden for females.

As seen from Table 4, the most common tumors were those associated with the endocrine system and comprised pituitary adenomas (which were more common in females), benign adrenal pheochromocytomas (largely in males), and thyroid C-cell adenomas. Other common tumors were those of the integumentary system comprising mammary gland fibroadenomas, adenocarcinomas, and adenomas (in females) plus skin fibromas/dermal fibromas, keratoacanthomas, and hair follicle tumors (largely in males). Common reproductive system tumors were testicular interstitial cell (Leydig) tumors and uterine stromal polyps; lymphocytic leukemia tumors in males were the most common tumors of the hematopoietic/lymphoreticular system. Remaining tumors were present at <5%.

Overall, slight differences in the number of individual tumors were only seen between the control groups (control I vs. control II) on a few occasions, most notably as follows:

As seen from Table 5, there was no specific pattern of incidence for less common tumors that, in the most extreme case, were only seen on 1 occasion among all the animals examined. In agreement with the overall variation between the 2 control groups, all 13 studies individually showed cases of “notable” differences as can be seen from Table 6, especially for benign adrenal pheochromocytomas among males and skin fibromas/dermal fibromas among both sexes or indeed, thyroid C-cell adenomas in males with an incidence of 13 vs. 5 in control I and control II, respectively. As noted in the Discussion, it is the magnitude of the difference with relevance to findings in drug-treated groups and in relation to background data that is important.

Discussion

This paper shows that, in general, no major differences occur for any tumor incidence between dual control groups across the full range of mammalian body systems when assessed in 13 in-house carcinogenicity studies over a period of 12 years. Interstudy variation occurred for some tumors but did not follow any particular pattern, although the incidence of benign adrenal pheochromocytomas in males was quite variable in a number of the studies. These results are interesting, as little has been published in this area. An evaluation of the Food and Drug Administration (FDA) and National Toxicology Program/National Cancer Institute (NTP/NCI) database showed that most rodent carcinogenicity studies had 1 control group and only 18 cases of duplicate control groups (Contrera et al., 1997). These latter studies appear to be a series of experiments performed with 18 color additives in the Charles River CD rat (Haseman et al., 1986). Interestingly, Contrera et al. (1997) reported that in 12 of these studies statistically significant differences were found for tumor findings between these groups.

However, this finding is not fully supported in the referenced Haseman et al. (1986) paper. The latter authors did indeed report marked study-to-study variability for certain tumors and found statistically significant (p < 0.05) paired control differences in 12 of the studies (see Table 7). However, when they analyzed these results to see if these findings exceeded chance expectation, no evidence of extrabinomial within-study variability between the 2 concurrent control groups was found. Indeed, the total number of observed significant paired-control differences was virtually identical to what would be expected from usual binomial model assumptions. The study authors also postulate that the marked study-to-study variability may be related to the fact that the studies were performed at 4 different testing laboratories and suffer from some histopathological deficiencies and inconsistencies.

Thus, from these study results and from the present reported studies, some interstudy variation can be expected in carcinogenicity study control group tumor incidences. In most cases, such variation does not cause an issue in assessment for potential carcinogenic risk. However, in those occasions where a low or high incidence of a tumor finding appears in a control group (as can occur even between concurrent control groups as indicated by e.g., 13 vs. 5 tumors for thyroid C-cell adenomas in 1 study and 0 vs. 3 or more tumors on various occasions in other studies), then a value for dual control groups can be argued when, for example, interpreting an increased incidence of the tumor in the high drug-treated group. The fact that such differences can be statistically significant is in itself not necessarily key; what is important is the magnitude of the difference in conjunction with factors such as preneoplastic/hyperplastic changes, evidence of increasing tumor incidence with increasing dose and background data incidence.

Thus, in the case of adrenal pheochromocytomas, which has an incidence of 7/60 and 14/60 for the 2 control groups in 1 study, an incidence of 14/60 in the high drug-treated group could easily be discounted as nontreatment related, although it could also be argued that background data would have allowed a similar conclusion. In a recent study in our laboratories, malignant lymphomas were seen at an incidence of 3, 8, 3, 4, and 7 (males) and 7, 22, 4, 8, and 20 (females) in control I, control II, low, mid, and high dose groups, respectively. In the absence of control II, the high tumor incidence seen in high-dose females plus the statistically significant dose trend could have resulted in the conclusion of evidence of a drug-induction for this tumor. The use of dual control groups to support interpretation of rare tumors is also valid, although results from the presented 13 carcinogenicity studies indicate that such tumors were often seen in both control groups anyway. Overall, in all these cases, scientific assessment would involve a careful evaluation of tumor incidence across all study groups (control and drug-treated) with reference to contemporary mean and/or overall range of tumor incidences. Indeed, it has been pointed out elsewhere that 2 control groups within the same study can occasionally show notable differences, stressing the importance of knowing the range of incidences rather than just mean incidence (Gopinath, 1994). Results from the present investigation show that the continued use of dual control groups in carcinogenicity studies has a role in the assessment of tumorigenic risk.

The literature contains a number of papers reporting survival and/or spontaneous neoplasm incidence for Sprague–Dawley rats (e.g., Chandra et al., 1992; McMartin et al., 1992; Keenan et al., 1995; Attia, 1996; Knight et al., 1996; Kaspareit and Rittinghausen, 1999; Hooks and Harling, 2001; Britton et al., 2004; Son and Gopinath, 2004; Tennekes et al., 2004). Chandra et al. (1992) showed survival of 42–65% (males) and 41–53% (females) from 17 carcinogenicity studies in the late 1980s/early 1990s while McMartin et al. (1992) reported values of 33–43% (males) and 27–44% (females) from a survey of 9 in-house studies conducted between 1984–1991. Further authors report survival of 44% (males) and 32% (females) from a range of studies in the period 1993–2000 (Hooks and Harling, 2001) plus contemporaneous survival values of 39–40% (males) and 33–39% (females) (Britton et al., 2004). These values compare with those of 32–60% (males) and 22–57% (females) seen in the present reported studies.

The present investigation shows that the majority of tumors were benign in nature and the incidence of malignant forms was 12–15%; this value compares well with the 17–19% incidence reported elsewhere (Chandra et al., 1992). This paper found pituitary adenomas to be the most frequent neoplasm at 48–49% (males) and 70–75% (females). These values are higher than reported by Chandra et al. (1992), namely 27% (males) and 49% (females) and by Kaspareit and Rittinghausen (1999), namely 20% (males) and 39% (females) but are less than the frequency reported by McMartin et al. (1992), namely 62% (males) and 85% (females). Attia (1996) reports a pituitary adenoma incidence of 47% and 62% for males and females, respectively, and recently values of 40% and 71% (Son and Gopinath, 2004) and 51–52% and 72–73% (Tennekes et al., 2004), respectively have been cited.

The next most common tumors in male rats were skin fibromas/dermal fibromas (20–25%), benign adrenal pheochromocytomas (12–18%), thyroid C-cell adenomas (12–15%), skin keratoacanthomas (9–13%), skin benign hair follicle tumors (3–7%), lymphocytic leukemia tumors (5%), and testicular interstitial cell tumors (4–5%). In females, the next most common neoplasms were mammary fibroadenomas (56–58%), mammary adenocarcinomas (11–14%), thyroid C-cell adenomas (9–11%), uterine stromal polyps (6–7%), and mammary adenomas (5–6%). This pattern of tumor findings and incidences generally compares well with those seen in other studies in Sprague–Dawley rats (e.g., Chandra et al., 1992; McMartin et al., 1992; Attia, 1996; Kaspareit and Rittinghausen, 1999) although the incidence of skin tumors in males is somewhat higher than reported in other studies in this strain. Values for less common tumors also compare well with those given in these papers.

The general similarity of tumor incidence in the present study to those reported elsewhere for Sprague–Dawley rats is comforting bearing in mind differences in study design. Such differences include Sprague–Dawley strain/supplier, diet (composition and amount), and caging (single vs. gang housed). Discussion of how these factors can affect tumor incidence is outside the goal of this publication but is well covered in the literature (e.g., Hardisty, 1985; Keenan et al., 1995; Greim et al., 2003; Haseman et al., 2003).

It has been highlighted that historical control data has a role in the evaluation of rare neoplasms, high-incidence tumors, marginal increases in incidence and in the assessment of the quality of a study (Greim et al., 2003). However, it is known that variation of incidence of tumor types within the same strain and sex and, within the same laboratory, occur due to the progression of time (Gopinath, 1994). In order to avoid such “genetic drift” when using historical data, it has been suggested that only studies performed during the last 5 years prior to the new study, should be accessed (CPMP/SWP/2879/00). Others have recommended that historical comparisons be limited to control data from the previous 3–4 years of the new study (Haseman et al., 1984; Fairweather et al., 1998). The NTP uses a historical control database with a time window of 7 years (Haseman et al., 1998).

From an analysis of certain tumor types over a period of 9 years, Gopinath (1994) showed that although there were fluctuations, incidence rates remained within a small band and no clear evidence of a time-related drift in rates was found. Recently, a review of the historical control data for 20 carcinogenicity studies generated in 1990–2002 in Sprague–Dawley rats showed little variability with time (Son and Gopinath, 2004). In a further evaluation of time-related changes in the incidence of spontaneous neoplasms in the adrenals, mammary gland, liver, pituitary, and pancreas of Sprague–Dawley rats from studies performed in 1986–1996, no obvious change was seen in the majority of tumor types (Tennekes et al., 2004). A small positive (increase with time) trend was seen for mammary gland fibroadenomas, while a small negative (decrease with time) trend was seen for pituitary adenomas (both sexes) and benign adrenal pheochromocytomas (males). An evaluation of tumor incidence using 4-year time points over the 12-year period in the present investigation has also shown no obvious difference in the number or pattern of tumors recorded. Standardizing tumor burden by incidence in the total number of animals assessed has not shown any strong evidence of drift although a slight trend for a lower incidence for males and for a higher incidence for females was seen.

In conclusion, this paper shows that no major differences in tumor incidences occur when dual control groups are used. However, occasional interstudy variation was seen for some tumors, indicating that the use of 2 control groups has a role in the interpretation of findings in drug-treated groups. Inter-study variation assists such interpretation when one control group, but not the other, mirrors elevated tumor incidence in one or more of the drug-treated groups. Thus, dual control groups give greater reassurance in assessing for biological significance of any increased tumor incidence. Such use also allows the buildup of contemporary background data at a greater rate.