Exp Toxic Pathol1999; 51: 523-536
URBAN & FISCHER
http://www.urbanfischer.de/joumals/exptoxpath
Bayer AG, Institute of Toxicology, Wuppertal, Germany
Trends in mortality, body weights and tumor incidences of
Wistar rats over 20 years
R. EIBEN and E. M. BOMHARD
With 27 figures and 5 tables
Received: October 20, 1998; Revised: January 21, 1999; Accepted: March 13, 1999
Address for correspondence: R. EIBEN, Bayer AG, Institute of Toxicology, D - 42096 Wuppertal, Germany.
Key words: Mortality, Wistar rats; Body weight, Wi star rats; Food intake, Wistar rats; Overnutrition, Wistar rats; Tumor
incidences, long-term studies; Carcinogenicity, genetic drift.
Summary
Historical data of more than 8.000 Wistar rats (designation: WISW SPF Cpb) used as controls in seventy 2-year
studies terminated between 1975 and 1994 were analyzed
for time trends in food consumption, terminal body weight,
mortality, and tumor incidences.
In males there was a significant (p < 0.01) time trend towards higher terminal body weight and a tendency (p > 0.05)
to lower incidences of pituitary tumors and adrenal pheochromocytomas, while mortality remained stable. Leydig
cell tumors showed a significant (p = 0.0005) positive trend.
In females, terminal body weight did not increase over time
but pituitary and mammary tumors showed a marked and
highly significant (p = 0.0001) increase, which explains a
significant (p = 0.0045) positive trend in mortality. There
was a significant (p =0.0001) negative time trend for uterine
adenomas/carcinomas and a slight tendency (p = 0.4135)
towards a decreased incidence of endo-metrial stromal polyps.
Since the average food intake data do not indicate a time
trend the changes observed might probably not be related to
higher caloric intake. In contrast to other authors we could
not find a positive correlation between either body weight
and incidences of pituitary tumors or body weight and mortality. Certain selection measures at breeding and/or a genetic
drift over time might explain the time trends observed. This
data does not yet indicate a need for a change in ad-libitum
feeding of these animals.
Introduction
In rodent bioassays historical background data of the
animal strain used and the knowledge about possible positive or negative trends in important parameters such as
mortality, body weight and frequencies of spontaneously
occurring tumors is necessary to distinguish between ap-
parent and real effects in carcinogenicity studies. While
reports on incidences/ranges of such data are numerous for
all more widely used laboratory rat strains (i.e. Fischer 344,
Sprague-Dawley, Wi star) rather few publications are available analyzing trends over longer time periods in this respect.
In one of these analyses RAO et al. (1990) demonstrated
for a group of altogether 5,184 male and 5,289 female
Fischer 344IN rats a significant trend in males towards lower
survival rates within a period of 11 years (1970-1981).
They related this trend to diseases associated with increasing
body weight, prevalence of leukemia and changes in the
criteria for euthanasia of moribund animals. The time trend
for the survival of females, however, was not significant.
In addition, there were highly significant positive time trends
of leukemia, anterior pituitary and thyroid C-cell tumors
in both sexes, adrenal pheophromocytomas in males and
mammary tumors and endometrial stromal polyps in females. The prevalence of mammary tumors in females
and pituitary tumors in males had a highly significant positive association with body weight. A similar analysis of
2-year bioassays is available for studies with SpragueDawley rats, which had been started between 1979 and
1990 and comprised a total of 2,140 control animals of
two different variants (NOHYNEK et al. 1993). One of these
variants [Crl: COBS-CD (SD)BrJ was used in the years
1979-1985, the other [Crl: COBS-VAF-CD (SD)BrJ being
employed in the years 1988-1990. While, within these
two periods, there were no noticeable time trends with regard to body weights at the different time-points of study
and mortality, a striking difference between the two variants
concerning these two parameters was seen: the VAF variant,
consuming more food, showed markedly and significantly
increased body weights and lower survival rates. An exemplary comparison of the pathology data of these two strains
conducted for some single studies showed an increase in
0940-2993/99/51/06-523 $ 12.00/0
523
the incidence of pituitary tumors in males, mammary
fibroadenomas in females, and an increase in the incidence of glomerular nephrosis.
Recently, the relationship between increased body weight,
reduced life expectancy as well as increased rates of specific tumors and non-neoplastic age-related changes has
been confirmed by numerous authors for the Fischer 344
rats (GRIES and YOUNG 1982; HASEMAN et al. 1992, 1997,
1998; RAo et al. 1990; ROE et al. 1995; Ross et al. 1970;
SEILKOP 1995; TURNBULL et al. 1985).
Especially the experience that it was impossible to conduct a number of studies over the required minimum test
period of 24 months startled the study directors in charge
and led to the start of numerous investigations looking for
appropriate measures how to prolong the life-span of these
strains again. (KEENAN 1998; KEENAN et al. 1998; CHRISTIAN
et al. 1998; HART et al. 1995; SINHA et aI. 1988).
In this respect, no problems have so far been described
for the Wistar rat. From the data of our own studies conducted between the years 1973 to 1989, no trend towards
an increase in mortality was seen (BOMHARD et al. 1986;
BOMHARD and RINKE 1994). Thirty-month studies (study
starts between 1979 and 1985) could also be carried out
without problems and resulted in mortality rates of about
50 % on average at the end of study (BOMHARD 1992). Since
no specific longitudinal analyses of the parameters being
discussed here were performed in these papers and a larger
data base is now available for the following years, the
study in hand intends to answer the following questions:
1. Do time trends exist in the Wistar rat for such parameters
as survival, body weights, food intake, overall tumor
rates and specific tumors?
2. If the answer is yes, are they comparable with those in
other rat strains?
3. Are they possibly so pronounced that it is advisable to
take intervening measures (such as dietary restriction)?
Material and methods
Experimental animals, housing conditions and nutrition
of the animals: Wistar rats (WISW SPF Cpb) used were SPF
bred and supplied by Harlan-Winkelmann, formerly Winkelmann, Borchen, Germany. During the years mentioned no
other rats were brought into Winkelmann's breeding rooms.
The original breeding pairs (Cpb: WU) of WINKELMANN were
derived from ZEIST, The Netherlands, in 1970. At first the
strain designation was Wistar TNOIW70, after a hysterectomy
in 1974 it was WistarTN01W74. From January 1981 the strain
designation has been BOR:WISW(SPF Cpb). At beginning
of the study the rats were about 5-7 weeks old.
The rats were housed individually, under conventional
conditions, in Makrolon® type II cages (SPIEGEL and GONNERT 1961) on low-dust, soft-wood shavings supplied by
Ssniff GmbH, Soest. The room temperature was adjusted to
22 ± 2 0c. The humidity was set up to 55 ± 10 %. An artificiallightldark cycle of 12 hours was used. From July 1991
onwards rats were kept in animal rooms located within a barrier system. Additionally, at that time a larger Makrolon®
cage (type IIa) was introduced for individual animal housing.
Table 1. Certified specification of nutrients in altromin®1321 (meal)/1324 (pellet)
diet used 1972-1994.
Published by Altromin in
1971
1978
1982
1989
Crude Protein %
Crude Fat %
Crude Fibre %
Ash %
Moisture %
Nitrogen Free Extract %
Metabolizable Energy MJ/kg
19.0
4.5
6.0
8.0
12.0
19.0
4.0
6.0
7.0
13.5
50.5
11.9
19.0
4.0
6.0
7.0
13.5
50.5
11.9
19.0
4.0
6.0
7.0
13.5
50.5
11.9
*
12.1
* no data available
Table 2. Mortality, body weights and food intake established in Wistar rats at an age about to 25 months.
Males (n = 4063)
Mean
Min.-Max.
Cumulative Mortality
Terminal Body Weights
Mean Food Intake per Rat per Day
(averaged over 24 months)
524
Exp Toxic Pathol51 (1999) 6
(%)
(g)
(g)
=
Females (n 4068)
Min.-Max.
Mean
16.6
423
2- 37
381-476
21.8
264
10- 40
228-304
19.2
15- 23
15.4
13- 18
Table 3. Selected#) tumor types in Wistar rats from 2-year studies.
percentage incidence2)
males
sex
females
organ I tumor type!)
&)
mean
min.-max.
mean
min.-max.
Pituitary, pars distalis
- Adenoma
- Adenocarcinoma
- Adeno(carino)ma*)
b
m
blm
15.5
0.2
15.7
2-38
0-4
2-38
28.8
0.3
29.0
7-58
0- 5
7-58
Adrenal glands
- Cortical adeno( carcino )ma
- Medullary tumor*)
blm
blm
1.3
8.2
0-14
0-30
1.2
1.4
0-10
0-8
Thyroid glands
- Follicular cell tumor*)
- C-cell tumor
blm
blm
1.3
7.6
0-6
0-22
0.8
6.7
0- 6
0-28
Pancreas
- Islet cell tumor
blm
1.0
0- 4
0.3
0- 2
0-10
0-32
0-22
0-50
Mammary glands in females
- Adenoma
- Fibroadenoma
- Adenocarcinoma
- Total mammary tumors*)
blm
1.0
6.8
3.3
11.1
Ovaries
- Granulosa/theca cell tumor*)
blm
1.7
0-10
11.9
0.8
5.7
6.5
0-45
0-10
0-20
0-24
b
b
m
Uterus (incl. cervix)
- Stromal polyp (incl. NOS)§)*)
- Adenoma
- Adenocarcinoma
- Adeno(carcino)ma*)
blm
Testes
- Leydig cell tumor*)
blm
7.0
0-22
Hemolymphoreticular system
- Malignant lymphoma
- Histiocytic sarcoma*)
m
m
0.8
0.6
0- 4
0- 8
0.4
0.3
0- 4
0- 4
Vascular system/mesenteric lymph nodes
- Hemangio(sarco)ma*)
blm
1.6
0-24
0.5
0-10
Vascular system/other locations
- Hemangio(sarco)ma
blm
0.4
0- 2
0.5
0- 4
b
b
m
Liver
- Hepatocellular adenoma
- Hepatocellular carcinoma
- Hepatocellular adeno( carcino)ma
- Cholangio(carcino)ma
b
m
blm
blm
0.2
0.5
0.7
0.1
0-2
0- 6
0- 6
0- 2
0.5
0.2
0.6
0.4
0-6
0-2
0- 6
0- 6
Kidney
- Lipomatous tumor
blm
0.2
0- 4
0.2
0-4
Urinary bladder
- Transitional cell tumor
blm
0.1
0- 4
0.0
0- 2
#) occurring with a frequency ~ 4 % in any of the 70 studies; *) frequency of this tumor type evaluated by a longitudinal
analysis (see figures); §) not reported in the first 11 studies; &) b = benign, m = malignant; NOS = not otherwise specified;
I) tumors found in paired organs were counted as one tumor; 2) rounded
Exp Toxic Pathol 51 (1999) 6
525
Animal rooms and all cage furniture were cleaned in most
cases weekly otherwise at least four weekly. The diet consisted of certified Altromin® 1321 meal or Altromin® 1324
pellets (3 studies only), which were provided for ad libitum
feeding. From October 1988 onwards 1 % peanut oil was added
to the powdered diet to avoid dust formation. The specification (table 1) ofthe nutritional ingredients of the diet was
essentially the same during the whole period.
Food intake was measured weekly. Body weights were
measured weekly (first 6 months) or bi-weekly thereafter.
Tap water was given to the animals ad libitum.
Necropsy and histological technique: Rats dying intercurrently or killed in moribund conditions were necropsied as
soon as possible and subjected to gross pathological evaluation. Selected organs and all neoplasms deemed evaluable
were fixed in buffered formaldehyde solution.
All rats alive at termination after a study period of24 months
were anesthetized with diethyl ether, killed by exsanguination and investigated macroscopically. Organs as recommended by the guidelines for chronic toxicity studies being
in force were fixed as described above. The fixed organs and
tissues were embedded in paraplast, cut into sections of
about 5 microns thick and stained with hematoxylin and
eosin. The bones were decalcified using EDTA. In some cases
special fixatives and/or staining methods were used, usually
to clarify tumor diagnosis.
The tumor nomenclature employed for tabulation relies
as far as possible on proposals by MOHR et al. (1990).
Data and evaluation method: The data evaluated originate
from 70 male and female groups (each 50 to 100 animals)
used as control groups in chronic rat studies completed at
Bayer AG Toxicology in the years between 1975 and 1994.
For each of the 70 male and female groups cumulative mortality rate, terminal body weight, mean food intake (averaged over 24 months) as well as incidences (as rounded percentages) of frequent tumors, overall tumors, and tumorbearing animals were calculated. This data was the basis for
mean value calculations (tables 2-4), which were then used
for a longitudinal analysis done by plotting means against
the time point the study was terminated. In cases where a positive or negative trend was evident in males and/or females
a diagram showing the regression line calculated over the
whole 20-year period by the EXCEL 5.0 program is given.
In order to discern positive or negative time trends each regression line was evaluated statistically applying of Students
t-test by using SAS 6.12 on a Windows NT 4.0 Workstation
platform (WEISBERG 1980). Multiple test corrections were
not taken into account.
Tumor types with a frequency below 4 % in any of the
male or female groups are not described in detail, but taken
into account when incidences of overall tumors and of
tumor-bearing animals were evaluated.
If appropriate, benign and malignant tumor types such as
adenomas and adeno-carcinomas were combined according
to MCCONNELL et al. (1986). Only primary tumors were taken
into account.
Animals or organs that could not be investigated histopathologically due to autolysis were not considered in the
evaluation. Tumors of the same type occurring in paired organs as well as several tumors of the same type in one organ
were considered only once in the evaluation.
Some of the tumor and mortality data used has been published previously (BOMHARD et al. 1986: data from studies
terminated 1975-1976; BOMHARD and RINKE 1994: data from
studies terminated 1977-1982). Data from studies terminated
between 1983 and 1994 was taken from unpublished internal
reports.
Results
Mortality, body weight, food intake and tumor data of
4063 male and 4068 female Wi star rats from the control
groups of studies from the last 20 years were evaluated.
All mean data including the variation range mentioned in
this chapter is summarized in the tables 2-4.
Mortality, body weights, and food intake
Mortality: During the period indicated a mean cumulative
mortality of 16.6 % was calculated for males and 21.8 % for
females, each with some variation between the studies. In
males no significant increase in mortality rates is visible over
time, however, there were relatively low rates around the
year 1985, whereas in females the regression line constantly
increases significantly (p = 0.0045) with time (figs. 1-2).
Terminal body weights: Over this period female rats remained relatively stable (p > 0.05) in mean terminal body
weight. In contrast, males showed a significant (p = 0.0001)
trend towards higher body weights (figs. 3-4). The mean
terminal body weight was 423.0 g for males and 264.2 g
for females.
Table 4. Incidence and total number of tumors and tumor bearing Wistar rats.
Males
Females
Total Number of Tumors
Mean Incidence of Tumors
Incidence Range of Tumors
%
%
2169
56.3
6-120
2745
69.4
26-142
Total Number of Rats with Tumors
Mean Incidence of Rats with Tumors
Incidence Range of Rats with Tumors
%
%
1691
41.6
6-68
2089
53.2
24-82
4063
4060
Number of Rats Examined
526
Exp Toxic Patho151 (1999) 6
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Figs. 1-6. Mortality, terminal body weight and food intake data from control groups of70 two-year studies with Wistar rats
terminated between 1975 and 1994. Mortality and mean body weights at study termination (week 104) were plotted against
the time point when the study was terminated as were food intake data (g/animal /day) averaged over the whole study period.
Food intake: The longitudinal analysis of food intake data
(figs. 5-6) revealed no remarkable trend neither in males
nor in females. The mean food intake calculated per rat and
day was 19.2 g for males and 15.4 g for females. There
was no remarkable difference in body weight or food intake
between corresponding groups fed pellets or meal diet.
Wistar rats. Most tumors occurred in endocrine and reproductive organs. For reasons of comprehensibility the
evaluation of tumor data was limited to selected types (details see under Material and methods).
Incidences of selected tumor types
Pituitary: Tumors of the pars distalis of the pituitary were
the most frequent tumor types in both male and female rats.
Nearly all ofthem were classified as benign (table 3). Pituitary tumors occurred in each of the groups and were
noted generally with a higher incidence in females than in
Benign and malignant tumors occurred in various organs
with more or less wide variation. The overall tumor spectrum was roughly in line with the tumor profile known for
Endocrinium
Exp Toxic Patho151 (1999) 6
527
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Figs. 7-14. Incidences of selected tumor types in the endocrinium of control groups of 70 two-year studies with Wistar rats
terminated between 1975 and 1994. Group incidences (%) of pituitary adeno(carcino)mas, adrenal medullary tumors, thyroid follicular and C-cell tumors were calculated for each of the 70 studies and plotted against the time point when the study
was terminated.
528
Exp Toxic Patho151 (1999) 6
males. A mean frequency of 15.7 % and 29.0 % was calculated for males and females, respectively. Over the time
pituitary tumor incidences decreased slightly (p = 0.0953)
in males, but increased markedly (p = 0.0001) in females
(figs. 7-8). During the last five years they occurred with
about two-fold higher rates in females than during the first
five years.
Adrenal glands: Cortical adenomas/carcinomas in adrenals occurred in male (1.3 %) and female (1.2 %) rats with
relatively low incidences on a constant level (graph not
shown) over the twenty years. Only 2.2 % (males) and 8.9 %
(females) of them were malignant. In male groups this
tumor was seen with a frequency of 1 to 8 % (25 cases).
One extreme value of 14 % was noted in 1981. Among
the female groups cortical tumors were observed four
times in 8 to 10 % of the rats, whereas in 21 cases the incidences varied between 1 to 6 %.
Medullary tumors, mostly of the pheochromocytoma
type were seen in males (8.2 %) more frequently and with
higher variation than in females (1.4 %). About one tenth
of these were malignant (males 10.2 %; females 10.6 %).
There was no significant time trend in males and females
(figs. 9-10).
Thyroid glands: Benign or malignant follicular cell tumors
were observed relatively seldom with a mean frequency
of 1.4 % in males and 0.8 % in females. Of these 18.5 %
in males and 8.3 % in females were classified as malignant.
In most of the groups follicular cell tumors occurred with
a frequency between 0 and 2 %. Higher incidences (3-6 %)
were noted in very few groups between 1975 and 1991 in
males and between 1975 and 1985 in females, but not thereafter. Thus, a somewhat decreasing trend is apparent during
the last years for both sexes (figs. 11-12), however, without statistical significance.
C-cell tumors ( = parafollicular tumors) occurred with
higher incidences in males (7.6 %) and females (6.7 %)
than follicular cell tumors. Among them 23.8 % (males)
and 10.3 % (females) were found to be malignant. In males
a C-cell tumor was recorded 62 times with a frequency of
2-16 % and only in two groups with higher rates of 20 to
23 % (1982 and 1989). Considering the first and last fiveyear period, males necropsied in 1989 and later showed
slightly enhanced incidences (fig. 13). Over 20 years, however, no statistical significance exists. In 57 female
groups 2 to 15 % of the rats had developed C-cell tumors.
Higher incidences (18 to 28 %) were found in four cases
(all between 1977 and 1982). The time trend is again not
significant (fig. 14).
Pancreas: The average incidence of benign or malignant
islet cell tumors was 1.0 % in males and 0.3 % in females.
Twenty-eight male and 12 female groups exhibited islet
cell tumors with a frequency between 1 and 4 % or 1 and
2 %, respectively. There was no time-dependent trend. The
ratio between benign and malignant tumors was 87.8 %
to 12.2 % in males and 92.3 % to 7.7 % in females.
Reproductive organs and female mammary gland
Female mammary glands: Adenomas, fibroadenomas
and adenocarcinomas of the female mammary gland occurred with a frequency of 1.0, 6.8, and 3.3 %, respectively. There is a highly significant positive trend (data
not shown) in all three case. The overall rate of these
tumor types is 11.1 %, which was in the last five years
four times higher than during the years between 1975 and
1979. Thus, a marked increase (p = 0.0001) in overall
mammary tumor incidences including two maximum values 48 and 50 % in the year 1993 is evident (fig. 15).
Ovaries: Ovarian granulosa-theca-cell tumors (benign:
89.7 % or malignant: 10.3 %) were seen with a mean frequency of 1.7 %. As demonstrated in figure 16 a slightly
negative, though not significant trend is obvious for this
tumor type from the year 1981 onwards.
Uterus: Among the uterine tumors, stromal polyps (not
consequently evaluated as tumor before 1976) occurred
with the highest frequency (11.9 %) followed by adenocarcinomas (5.7 %) and adenomas (0.8 %). As can be seen
from figure 17, the stromal polyp rate showed a slightly
negative tendency during the years 1988 and 1994. A more
prominent (p = 0.0001) decrease is visible for the combined
incidences of adenomas/adenocarcinomas (mean value
6.5 %) from the year 1975 onwards with the exception
that there was a relatively high rate of 24 % in one group
necropsied in 1983 (fig.18). This negative trend is based
on the fact that between 1987 and 1994 more groups
lacking uterine adenomaslcarcinomas were found than between 1975 and 1986.
Testes: The mean rate of benign (99.6 %) and malignant
(0.4 %) Leydig cell tumors was 7.0 %. The relatively high
variation range of this tumor increased with time. There
is a significant (p =0.005) ascending time trend with maximum values of 22 % in 1990 and 1993 (fig. 19).
Other organs
Hemolymphoreticular system: In the hemolymphoreticular system malignant lymphoma prevalence was low:
0.8 % on average in males and 0.4 % in females. The incidence was 1-2 % in 18 male and 13 female groups, and
4 % in five male and two female groups. Over the time no
remarkable trend could be detected neither in males nor
in females. Histiocytic sarcomas were observed nearly as
frequent as malignant lymphomas (0.6 % in males and
0.3 % in females), but showed a positive trend (p < 0.01
in both sexes) starting around 1990 in males and in females
(figs. 20-21). The peak incidences of 8 % (males) and 4 %
(females) were in the same study terminated in 1993.
Vascular system: Angiomatous tumors (hemangiomas,
hemangiosarcomas) were found in various organs with a
Exp Toxic Pathol51 (1999) 6
529
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Figure 17
Figure 18
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20
15
10
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•
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11175
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-.
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11180
Figure 19
11190
~
Figs. 15-19. Incidences of selected tumor types in reproductive
organs and female mammary gland of control groups of 70 twoyear studies with Wistar rats terminated between 1975 and 1994.
Group incidences (%) of mammary adeno( carcino)mas and fibroadenomas, of granUlosa/theca cell tumors, uterine stromal polyps,
uterine adeno(carcino)mas and Leydig cell tumors were calculated
for each of the 70 studies and then plotted against the time point
when the study was terminated.
total incidence of 2.0 % in males and 1.0 % in females. If
specific locations are considered, a significantly (p =0.003)
increased incidence was observed in the mesenteric lymph
nodes of males from 1981 onwards, but was not seen in
females (figs. 22-23). There was one male and one female
group (both in 1986) showing a relatively high incidence
of 24 % and 10 %, respectively. Angiomatous tumors of
the uterus, spleen, lungs, as well as in subcutis or mesentery occurred in 13 male and 8 female groups with a frequency of 1-2 %. Higher frequencies (3-4 %) were noted
in five female groups only.
Liver: Hepatocellular adenomaslcarcinomas (males: 0.7 %;
females: 0.6 %) and cholangiomas/cholangiocarcinomas
(males: 0.1 %; females: 0.4 %) were found with relatively
low incidences. There were 47 (male) or 50 (female) groups
530
Exp Toxic Pathol 51 (1999) 6
without any hepatocellular tumor, whereas in 19 groups
of both sexes this tumor type occurred with an incidence
of 1-2 %. In four male and one female group(s) rates of 4 to
6 % were observed, however, without a noticeable trend.
Cholangioma/cholangiocarcinoma prevalence was 1-2 %
in four male and 10 female groups, and 4 to 6 % in two
female groups without a noteworthy time trend.
Kidneys: Benign or malignant lipomatous tumors were
seen with a mean incidence of 0.2 % in both males and females. In six male and three female groups the frequency
was 1-2 % whereas it was 4 % in one male and two female groups. A time-dependent trend is not visible.
Urinary bladder: Transitional epithelial tumors occurred in only two male and one female group(s) (at 1 % and
10
"
10
8
a
6
S
•
0
1975
•
••
• •
2
••
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11180
1
1175
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0
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11180
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19115
Figure 21
Figure 20
25
:25
20
20
15
IS
10
10
..
5
11180
1
lymph_oI
1-.gioC~1n
01
lymph
0
11175
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5
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19115
0
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Figure 22
FemoIIe
~
1180
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Figure 23
Figs. 20-23. Incidences of histiocytic sarcomas and mesenteric lymph node hemangio(sarco)mas in control groups of70 twoyear studies with Wistar rats terminated between 1975 and 1994. Group incidences were calculated for each of the 70 studies
and then plotted against the time point when the study was terminated.
4 % in males, 2 % in females). Two transitional epithelial
tumors were reported for one female group only.
Other locations: In the remaining organs/tissues tumors
occurred with incidences of less than 1 % lacking any
trend.
Incidences of overall tumors and
tumor bearing animals
Overall tumors: The overall tumor incidences were 56.3 %
in males and 69.4 % in females and varied over a relatively wide range (table 4). The longitudinal analysis
(figs. 24-25) revealed a clearly ascending (p = 0.0001)
trend in females as a consequence of increasing pituitary
and mammary tumors. In males the tendency toward higher
tumor rates was less pronounced (p =0.0061).
Tumor bearing animals: Among the 8131 rats 41.6 %
males and 53.2 % females had developed a primary tumor.
The study to study variation is again high. As shown in
figures 26-27, the number of tumor bearing rats increases significantly over time more pronounced in females (p =0.0001) than in males (p =0.0053).
Discussion
Recent experience, which was confined to Fischer 344
and Sprague-Dawley rats, showed in some cases quite
dramatic time trends within certain rat strains/stocks. Basicall y, the following trends were described: increased food
intake, increase in body weights, reduction of the lifespan, increase in the incidence of diverse tumors. Both
outbred (Sprague-Dawley rats) and inbred strains
(Fischer 344 rats) were affected in this respect. Considerable effort was made, especially in the USA, to counteract these trends. The measures taken aimed at reducing
food intake, thus retarding body weight development, diminishing the frequency of tumors and other age-related
diseases and, finally, achieving a longer survival time for
rats. Since the proposed measure of food intake reduction
represents a significant interference with study conditions
and in all likelihood leads to considerable additional expenditure, while the total historical data base becomes virtually useless, we thought it absolutely essential to make
a longitudinal analysis of the Wi star rats used at our institute for more than 20 years. One special advantage was
the fact that diverse important general conditions had
remained almost unchanged for all studies within that
period.
Exp Toxic Pathol51 (1999) 6
531
"
11Or----
110.-------------------------------,
120
120
110
o~------+-------+_
lG7S
1
'II
__ __
~
20
.'. .
...-
•••
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:
•
•
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v..
100.-------------------------------,
"
t
. ...
eo
40
I •• ".
."
o~------~------+-------+_
19110
1875
••• $
Figure 25
,.
eo
.
•
1875
100.-------------------------------,
10
~:_~--~~~~~.~.~~'~'~,·~·T:.~~.
.... .•
'.
~
;("*
...
o~~----~~----+_------+_----~
+_--_+~
Figure 24
..
11190
____
20
1995
....
:r
,
0
1875
~
Figure 26
... ...
1_
..
..
\
'
.
e
:'
II1!1O
-..
..
• 't
1l1li5
Figure 27
Figs. 24-27. Incidences of the total numbers of tumors and tumor bearing rats of control groups of70 two-year studies with
Wistar rats terminated between 1975 and 1994. Incidences were calculated for each of the 70 studies and plotted against
the time point when the study was terminated.
This longitudinal analysis yielded important results for
a large number of parameters, which are presented hereinafter and compared with the experience of other authors.
Mortality, body weight, and food intake
Despite a slight but steady increase in the terminal body
weights of male groups, no effect on mortality was observed. It is, however, to be noted that the terminal body
weight increase within this period was less than 10 % and
that the body weight range was in any case by far lower
than that known for most Sprague-Dawley stocks (see
below). Since there was no time trend towards an increase
in the food intake, the possibility exists that the food utilisation of males has improved in this period (e.g. by betterfood digestibility). Another possibility, however, would
be different food intake at different ages (which was not
analysed in more details). A stable food consumption
during each of 10 carcinogenicity studies terminated between 1988 and 1993 and over the period in question was
also reported for Crl:CD BR Sprague-Dawley rats while
terminal body weight gain increased by 32 % in males
(from 421 g to 554 g) and by 26 % (from 284 g to 358 g)
in females (SIMONNARD et al. 1994).
532
Exp Toxic Pathol 51 (1999) 6
In contrast to males, females displayed an increase in
mortality over this period with virtually unchanged final
body weight and average food intake. The reason for this
increase is certainly the higher number of pituitary and
mammary tumors. Both tumor types are not primarily life
threatening as a rule, however, if they have grown to acertain size, the animals are usually terminated for animal
welfare reasons. The inverse correlation between body
weight and survival described by ROE et al. (1995) and
TURNBULL et al. (1985) for Wistar and Sprague-Dawley
rats (observed during some single experiments) cannot be
substantiated by this long-term comparison.
As far as body weight and mortality are concerned a
comparison with other authors' publications yields the
striking result that the Wistar rat used here differs considerably from most stocks of the Sprague-Dawley rat of different breeders. The terminal body weights of males are
mostly clearly higher than 600 g (ranges from circa 600
to 830 g), while the mean weights of females are more
than 450 g (ranges from circa 420 to 540 g) (NOHYNEK et
al. 1993; PETTERSEN et al. 1996; SIMONNARD et al. 1994).
Among the Sprague-Dawley stocks, however, there are
also some showing markedly lower terminal body weights.
The values for the Hsd:Sprague Dawley SD rat, for example, were 518 g for males and 324 g for females (PETTER-
SEN et al. 1996). In its mean terminal body weight the Wistar
rat is approximately comparable with the Fischer 344 rat.
For the latter, average terminal weights of 413 g for males
and 312 g for females for studies started between 1971
and 1977 were reported (CAMERON et al. 1985). In studies
initiated in the years from 1971 to 1981 the terminal body
weights of males rose from 408 g in 1971 to 480 g in 1981
(p < 0.001), those offemales from 307 g to 349 g (p < 0.001)
(RAO et al. 1990). Relatively high body weights can also be
reached by Wistar rats: after a study period of 104 weeks
the Wistar Crl: (WI) BR and Hla:(WI BR) rats supplied
by two American breeders showed mean body weights at
termination varying from 556 to 717 g in males, and 428
to 533 g in females (WALSH and POTERACKI 1994). There
were also marked differences between the two stocks:
Hla-rats weighed more at termination (males 686 ± 26 g;
females 504 ± 24 g) than Crl-rats (males 594 ± 34 g; females 438 ± 21 g).
With respect to mortality the rates of 20 % and about
25 % for Fischer 344 males and females, respectively,
during studies conducted in the 70s and at the beginning
of the 80s were very close to the rates determined for our
rats (CAMERON et al. 1985; RAO et al. 1990). Studies conducted in the following years revealed for the Fischer 344
rat a continuous increase in the mortality rate of animals
fed ad libitum (HASEMAN and RAo 1992; THURMAN et al.
1994). They were as high as 65 % for males and 41 % for
females in 2-year studies (THURMAN et al. 1994). High
mortality rates after 104-week studies are also reported
for the Crl:CD BR Sprague-Dawley rat: 64 % (range
57-67 %) for males and 66 % (range 56-73 %) for females
(McMARTIN et al. 1992) and 71 and 56 % on average for
males and females , respectively (PETTERSEN et al. 1996).
The figures for the Hsd:Sprague-Dawley rat, however,
are more favorable: 51 % for males and 37 % for females
(PETTERSEN et al. 1996). Such stock differences are also
evident in the Wistar rat. In ten bioassays conducted between 1980 and 1990, the average mortality of males and
females was 42 % and41 %, respectively, the ranges being
28-55 % for males and 26-56 % for females (WALSH and
POTERACKI 1994). In 13 2-year studies including 4,700
Wistar rats (no reference to the year of study performance,
presumably in the 70s), mortality at 24 months was even
approximately 70 % in males and 65 % in females (GRIES
and YOUNG 1982).
Since housing conditions in the different institutions
should be similar to a large extent (at least no significant
differences could be inferred from the publications) the
marked disparities between the various stocks of the same
strains might most likely be due to genetic differences.
Tumor data
Before analyzing and comparing time trends in tumor
data between the strain used at Bayer AG and those of
other labs, it seems sensible to compare the tumor incidences oflarger groups of Wi star, Fischer 344 and Sprague-
Dawley rats in carcinogenicity studies lasting for 2 years
(table 5). This presentation demonstrates that there are
partly quite remarkable differences between the individual
rat strains. Especially striking here are the very high incidences of Leydig cell tumors and (predominantly mononuclear cell) leukemias in Fischer 344 rats. The SpragueDawley rat shows a tendency towards high rates of pituitary
and female mammary gland tumors. Apart from that, the
table also shows a remarkable range of variation for the
diverse tumors within the individual strains. One reason for
this might be the influence of different diagnostic criteria
especially when assessing tumors of the endocrine system. But this does certainly not explain other, also striking
differences (e.g. in the mammary gland). Considering all
tumor types, the Wistar stock used by us is conspicuous,
despite a high survival rate, by the lowest incidences of
spontaneous tumors without a noteworthy reduction in
the tumor spectrum compared with the other stocks.
For the very frequent pituitary tumors (average incidence males: 15.7 %, females 29.0 %) and female mammary gland (11.2 %) tumors, a significant (p < 0.01) time
trend was observed in females. Highly significant (p < 0.001)
positive time trends for prevalences of anterior pituitary
tumors in both sexes and mammary tumors in females
have also been found in Fischer 344 rats (RAO et al. 1990).
While Fischer 344 males exhibited also a highly significant increase in pituitary tumors over time, Wi star males
used at Bayer AG showed a tendency (p =0.0953) towards
a decrease. Among the more frequent tumors there was a
significant (p < 0.0005) positive time trend for Leydig cell
tumors. A time trend for this tumor was not evaluated by
RAo et al. (1990), since almost all male Fischer 344 rats
may have such tumors. Among those tumors with rather
low incidences there were a few with a significant time
trend: histiocytic sarcomas in males (p = 0.0016) and females (p 0.0001), and vascular tumors in mesenterial
lymph nodes in males (p =0.003). Since RAO et al. (1990)
evaluated only common spontaneous tumors with a prevalence of approximately 10 % or more and these tumors
occur in the Fischer 344 rat only with a relatively low frequency, no comparable data is available. The only tumors
with a significant negative trend (p =0.00001) were uterine adenomas/carcinomas. Since these tumors as well appear relatively seldom in the Fischer 344 rat, there is no
published data available for a comparison. In Wistar rats
used by us no corresponding evidence was found for the
significant (p < 0.001) positive time trends observed in
Fischer 344 rats for prevalences of leukemia, anterior
pituitary adenomas in males, thyroid C-cell tumors in
both sexes, adrenal medullary tumors in males and endometrial stromal polyps in females.
=
Tumor data vs food intake, body weight,
and mortality
In literature, there are numerous references to correlations between higher body weight and tumor incidences,
Exp Toxic Pathol51 (1999) 6
533
Table 5. Incidences of selected tumors in Wi star, Sprague-Dawley and Fischer 344 rats.
Rat Strain
Source of Data
Males
Number of animals examined
in percent (%)
Terminal mortality
Pituitary: adeno(carcino)ma
Adrenals: cortical adeno(carcino)ma
Adrenals: medullary tumor
Thyroid: follicular cell tumor
Thyroid: C-cell Tumor
Pancreas: islet cell tumor
Testes: leydig cell tumor
Liver: hepatocellular
adeno( carcino)ma
Kidney: lipomatous tumor
Malignant lympoma
Lymph node: hemangio(sarco)ma
Histiocytic sarcoma
Females
Number of animals examined
in percent (%)
Terminal mortaJity
Pituitary: adeno( carcino )ma
Adrenals: corticaJ adeno(carcino)ma
Adrenals: medullary tumor
Thyroid: follicular cell tumor
Thyroid: C-cell tumor
Pancreas: Islet Cell Tumor
Mammary adeno( carcino)ma
Endometrial stromal polyp
Uterus: adeno(carcino)ma
Ovary: granulosa/theca cell tumor
Liver: hepatocellular
adeno( carcino )ma
Kidney: lipomatous tumor
Malignant tymphoma
Lymph node: hemangio(sarco)ma
Histiocytic sarcoma
Wistar
A
B
Sprague-Dawley
C
D
Fischer 344
E
F
4063
685
1340
585
740
1354
16.6
15.7
1.3
8.2
51
27.8
7.6
1.0
7.0
57.6
27.7
10.2
5.3
2.6
3.0
4.4
3.9
63.6
2.6
20.9
6.1
7.4
9.4
6.7
19-24
20.5
1.7
6.0
2.0
5.8
6.5
79.5
51
30.0
0.9
31.9
2.1
14.8
4.9
89.2
0.7
0.2
0.8
1.6
0.6
1.9
2.3
5.3
2.0
2.3
2.4
0.5
2.0
7.0
1.2
0.9
0.9
1.4
2.6
0.4
5.8
0.1
0.4
0.1
0.2
585
740
1351
23-28
30.3
1.5
0.4
39
53.1
1.9
5.1
0.9
13.6
2.1
46.3
14.2
1.3
1.3
4.8
1.5
4.1
3.8
0.9
4060
685
1329
21.8
29.0
1.2
1.4
0.8
6.7
0.3
11.1
11.9
6.5
1.7
59.1
54.9
10.5
1.6
1.6
4.4
2.0
42.8
9.6
1.6
2.2
54
49.3
2.2
0.8
1.0
3.3
31.3
2.4
0.2
0.3
0.6
0.2
0.4
0.5
0.3
3.3
1.2
4.2
0.7
1.0
0.6
0.3
1.6
0.1
1.0
1.1
88.1
5.1
6.2
2.9
7.6
4.9
54.6
10.0
0.5
0.2
6.0
0.3
1.0
1.2
1.3
5.1
0.1
14.6
14.1
0.7
0.3
0.3
0.4
1.1
1.1
1.4
0.1
0.3
0.0
0.1
A =rats used at Bayer AG (taken from table 3 of this paper); B = WALSH and POTERACKI (1994); C =CHANDRA
et al. (1992); D = McMARTIN et al. (1992); E = CHANDRA and FRITH (1992); F = HASEMAN et al. (1998);
- = data not available
the predominant conclusion being that increased body
weight enhances tumor risk (GRIES and YOUNG 1982; HASEMAN et al. 1992, 1997, 1998; RAo etal. 1990; ROE et al. 1995;
Ross et al. 1970; SEILKOP 1995; TURNBULL et aJ. 1985). The
tumor types most frequently mentioned in this respect
were pituitary tumors in males and females and mammary
tumors in females. This observation was made in all three
rat strains. The data presented in this paper does not con534
Exp Toxic Pathol 51 (1999) 6
firm these results. There are tendencies in opposite directions between body weight and pituitary tumors in males,
and while the body weight of females remained constant,
the pituitary and mammary tumors increased distinctly.
The question as to which factors contributed to these surprising results requires further analyses.
Due to the absence of changes in the food intake over
the period under investigation, the increase in diverse tu-
mors cannot be explained by higher caloric intake. High
caloric intake (determined mostly on the basis of increased
food intake in the course of 2-year bioassays in rats) was
in many cases held responsible for the rise in diverse
tumor incidences (KEENAN 1998; KEENAN et al. 1998;
CHRISTIAN et al. 1998; HART et al. 1995; SINHA et al.
1988). The fact that increased caloric intake and thus markedly elevated body weights not necessarily have to be
associated with more tumors is demonstrated by a longterm study in Sprague-Dawley rats receiving up to 30 %
D-glucose in the diet. In this study despite 16 % (males)
and 26 % (females) higher body weights and slightly increased food intake, no significant increase was observed
either in the number of pituitary (males and females) and
mammary tumors (females) or in the total number of
tumors. The only tumor type showing a significantly higher
incidence was the pancreatic islet cell adenoma in males
(BOMHARD et al. 1998). Though TURNBULL et al. (1985)
report about a significant (p < 0.05) positive correlation
between body weights and pancreatic islet cell tumors in
male Sprague-Dawley rats, they found markedly higher
correlations for pituitary (males and females) and mammary (females) tumors.
Concluding remarks
With respect to the question mentioned above our data
allows the following conclusions:
1. There are some time trends observed in the Bor: WISW
(SPF Cpb) Wi star rat concerning terminal body weight
(males), mortality (females) and some tumors.
2. The extent of these trends is lower than in other strains.
In some cases they are opposed to previous theories
about causal relationships.
3. They are not so pronounced that intervention appears
to be required. No necessity for resource-intensive measures such as dietary restriction can be inferred from
these results.
Although on the one hand the wish to improve the situation by such measures for the rat strains/stocks concerned and thus to permit "easy" performance of2-year bioassays seems to be understandable from the viewpoint of
the present Guidelines/Registration Requirements, there
are on the other hand also other options to be considered.
These could be for example:
a) changes in the breeding strategies away from fast growing to slowly growing animals;
b) diet optimization, e.g. by reducing the share of protein,
increasing the share of fibre;
c) reduction of the experimental period of cancer bioassays employing short-lived strains.
A reduced experimental period is already the current
practice with mice. Mice studies with short-lived outbred
strains (such as NMRI, CD-I, CF-l) are terminated in the
majority of cases after 18 months, those with the long-lived
hybrid strain B6C3Fl after 24 months as a rule. Considering
additionally that in the wake of the International Conference
on Harmonization (lCH) concrete deliberations have been
made and comprehensive validation studies are under way
with the aim of replacing the existing 18- to 24-months
mouse bioassay by models with a study period of presumably maximum 6 months (by using transgenic animals),
the desperate adherence to a study period of 2 years for
the rat bioassay seems at least worth for reconsideration.
The relevance of the results for humans should be and
remain the paramount objective of any study design modification. The doubts increasing over the last few years
about the benefits in relation to the present expenditure
(bioassays in two rodent species) could, in addition to the
problems described for diverse rat stocks, contribute to reconsidering the whole conception of the studies investigating carcinogenic effects.
Acknowledgements: The authors thank Dr. M. BECKA and
Dr. M. RINKE for their kind support and stimulating discussions. The assistance of Mrs. G. WEINAND in preparing the
manuscript is gratefully acknowledged.
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