Fd Chem. Toxic. Vol. 28, No. 3, pp. 157-167, 1990 Printed in Great Britain.All rights reserved

0278-6915/90$3.00+ 0.00 Copyright © 1990PergamonPresspie

CHRONIC TOXICITY/CARCINOGENICITY STUDIES OF SULPHAMETHAZINE IN FISCHER 344/N RATS: TWO-GENERATION EXPOSURE N. A. LITTLEFIELD*,W. G. SHELDONt, R. ALLEN$ and D. W. GAYLOR Department of Health and Human Services, Food and Drug Administration, National Center for Toxicological Research, tPathology Associates, Inc., National Center for Toxicological Research and ~:Computer Based Systems, Inc., National Center for Toxicological Research, Jefferson, AR 72079, USA (Received 22 August 1989; revisions received 8 November 1989) Abstract--Fischer 344 rats were given 10, 40, 600, 1200 or 2400 ppm sulphamethazine (SMZ) in the diet to determine the toxicity and potential carcinogenicity of SMZ. There were 225 rats of each sex in the control groups and 135 of each sex in each dose group. Animals were killed after 3, 12, 18 or 24 months of continuous dosing. Body weights, feed consumption, clinical observations, organ weights and histopathology data were collected. A slight decrease in body-weight gain was observed in the high-dose groups compared with the controls. No difference in feed consumption was found between the control and dosed rats. Mortality was inversely related to SMZ dose, especiallyin females, that is mortality was highest in the controls and decreased as the dose of SMZ increased. A statistically significantdose-related increase in the incidence of follicular cell adenocarcinomas of the thyroid gland was observed in the animals killed after 24 months. The incidences of non-neoplastic lesions of the thyroid gland in treated animals were significantly higher among treated animals than among controls; these lesions included follicular cell hyperplasia, follicular cell focal cellular change and multilocular cysts. The incidences of retinal atrophy, atrophy of the acinar pancreas (males), and dilatation of the uterine lumen also increased with increasing SMZ dose.

INTRODUCTION

Sulphamethazine (SMZ) [4-amino-N-(4,6-dimethyl2-pyrimidinyl)benzene sulphonamide; CAS no. 057-68-1] is an antibacterial sulphonamide drug used for prophylactic and therapeutic purposes in swine, cattle, horses, sheep, chickens, turkeys, dogs and cats. SMZ has been added to poultry and cattle feed and is effective for the prevention of atrophic rhinitis in swine. Sulphonamide residues in swine tissues attracted public attention when during 1977 the residues exceeded the 0.1 ppm tolerance level in the kidneys, liver and muscle of swine (Federal Register, 1970). Between 1974 and 1977, 12.4% of swine samples obtained by the FDA were in violation of these levels. In 1978, the withdrawal time (time drug administration is discontinued before the animal is slaughtered) was changed from 7 to 15 days to help prevent problems associated with residues in the meat (Federal Register, 1977). Swine fed non-medicated feeds have had residues above the 0.1 ppm tolerance level of sulphonamides when housed in pens containing urine or manure from swine previously fed sulphonamides. SMZ-induced dose-dependent thyroid hyperplasia has been observed in rats (Astwood et al., 1943; MacKenzie and MacKenzie, 1943; Swarm et al.,

*To whom all correspondence should be addressed at: HFT-140, Division of Comparative Toxicology, NCTR, Jefferson, AR 72079, USA. Abbreviations: SMZ = sulphamethazine; T3 = serum triiodothyronine; T4 = thyroxine; TSH = thyroid stimulating hormone. v ~ 2s/3-e

157

1973) but has not been found in man or in domestic animals when the drug was administered at therapeutic doses. Toxicological studies that were directed toward establishing 'no effect' levels were carried out under the auspices of the Animal Health Institute. SMZ had no effects at 2 mg/kg body weight in rats (unpublished data). In these studies, doses included 2, 6 and 20 mg/kg for 90 days. Hyperplasia of the thyroid gland was present in rats at 20 mg/kg and effects were equivocal in the animals fed 6 mg/kg. Hansen and Bichel (1942) conducted studies on selected sulphonamides, although SMZ was not included. One chemical, 6-sulphanilamido-4,6dimethylpyrimidine, which is structurally similar to SMZ, was implanted in the axilla space of 15 Wistar rats. One rat in the treated group developed an abdominal lymphosarcoma with metastases to the liver, another rat developed an adenocarcinoma of the kidney, while another rat developed a spindle-cell sarcoma in the spleen. SMZ, added at 0.2% in the diet for 35 to 42 days, caused atrophy of the testes, seminal vesicles and anterior prostate and follicular cell hyperplasia of the thyroid in rats. In hypophysectomized rats, only testicular atrophy was observed (Pan, 1948). Hypertrophy of the accessory organs has been reported along with precocious development of primary and secondary sexual characteristics in chicks. Accompanying these changes were decreased rates of blood clotting and haemorrhages, and a decreased growth rate (Asplin and Boyland, 1947). In a companion study to the study reported here, B6C3F, mice that were fed SMZ for up to 2 years, at doses of 300, 600, 1200, 2400 and 4800 ppm in the

N. A. LITTLEFIELD et al.

158

diet, had a dose-related increase in the incidence of follicular cell adenomas of the thyroid gland in both males and females (Littlefield et al., 1989). MATERIALS AND METHODS

Chemical. SMZ was purchased from American Cyanamid Company, Wayne N J, USA in one batch (Lot no. 3912) and was used as received. Highperformance liquid chromatography analysis of the SMZ indicated that the compound's purity was greater than 99%. SMZ was identified by mass spectrometry. Dosage preparation. The SMZ was administered in the feed continuously at doses of 10, 40, 600, 1200 and 2400 ppm. A separate group of animals was fed untreated feed and served as a control. The entire feed preparation process was conducted in a sanitized chamber. The feed was loaded into small labelled cardboard boxes by dose and packaged into stainlesssteel carrying cases for transport to the animals in the barrier rooms. Before mixing the diets, studies were done to determine the stability and extractability of SMZ from the feed, and to validate the analysis process. Extraction of the SMZ, when it was mixed directly into the feed, was difficult. Therefore, Poly Soy 20, manufactured by Ralston-Purina (a soyabean product that had been extracted with hexane and water) was used as a premix carrier. The SMZ was blended in the Poly Soy along with 4% (by weight) of mineral oil. The premix was then blended with Purina 5010M feed in 20.0-kg batches, using a 140-quart Hobart blender. The concentration of Poly Soy and mineral oil was kept constant in each dose mix, including the controls, at 7.75% (w/w) of the final product. Control diets were prepared in a different room using a 100-kg Marion blender. Samples of feed were analysed for dose certification before placing the feed in the animal cages. Specifications for the feed were established as + 10% of the target doses for the three high doses and + 20% for the 10 and 40 ppm dose mixes. Animal care and maintenance. Animals used in this study were from the F~a generation of dosed Fischer 344 rats (F344/N) that were bred in the National Center for Toxicological Research breeding barrier under specific-pathogen-free defined-flora conditions. The F0 weanling animals were randomly allocated to five dose groups under barrier conditions and both sexes were dosed at one of the five concentrations in their feed for at least 80 days. While still receiving medicated feed, the females were mated with males

(one male per female) of the same dose group. Brother/sister matings were avoided. Medicated feed remained in the cages at all times. Two males and two females were selected randomly from each litter (Fla generation) and allocated, three per cage as weanlings, to the 24-month study. The rats were usually weaned at ages 20-24 days. Littermates were not assigned to the same cage on the chronic study. The F~a animals continued on the same dose as their respective parents. Microbiological evaluations were conducted periodically on the rats, and on room air, floors, walls and cage litter every 2 wk. The drinking-water and the feed were checked for the presence of pathogenic organisms. Room temperature was controlled at 72 + 2°F and humidity maintained at 50 + 10%. A slight positive pressure with reference to the corridors was maintained in the animal room. Ventilation in the animal room was accomplished by 14 to 16 changes of air per hour; there was a 12-hr light/dark cycle. Hardwood chips were used as cage bedding. Feed, water, bedding and cages were changed weekly. Filter tops were used on all cages. Weekly weights of animals, feed consumption, clinical signs and other observations were recorded by a computer-supported program. The rats were identified by an ear-clip system and by cage cards that included experiment number, cage number, room number, dose code, rack number, start date and treatment number. Twicedaily checks were made and animals were removed from the experiment when they died or became moribund. All animals on the study received a complete autopsy and histopathological evaluation. Haematology and clinical chemistry analyses were conducted on samples taken at the 12- and 24-month kills. Experimental design. Groups of 135 rats of each sex were fed diets containing 10, 40, 600, 1200 or 2400 ppm SMZ for 3, 12, 18 or 24months (Table 1). Another group of 225 rats of each sex had no SMZ in their feed and served as controls. A total of 900 males and 900 females were used on the study. Only the thyroid gland was examined microscopically from the rats of the 3-month group. The animals were allocated as weanlings to the various groups and were continued on the diet of their respective dams after their allocation. Histopathology and clinical pathology. The following tissues were examined microscopically: adrenal gland, aorta, urinary bladder, bone marrow, brain, coagulating gland, ear, epididymis, oesophagus, eyeball, Harderian gland, heart, intestines (large and small), kidneys, lacrimal gland, liver, lungs, lymph

Table 1. Experimentaldesignof a chronicstudy in Fischer344/N rats fed SMZ for 3, 12, 18 or 24 months Time of autopsy (months) Total no. of rats Dose level (ppm) 0 (Control) 10 40 600 1200 2400 Total

Males 225 135 135 135 135 135 900

3

12

18

24

Females Males Females Males Females Males Females Males Females 225 15 15 15 15 15 15 180 180 135 15 15 15 15 15 15 90 90 135 15 15 15 15 15 15 90 90 135 15 15 15 15 15 15 90 90 135 15 15 15 15 15 15 90 90 135 15 15 15 15 15 15 90 90 900 90 90 90 90 90 90 630 630

Carcinogenicity of sulphamethazine nodes, mammary glands, skeletal muscle, sciatic nerve, ovaries, pancreas, nasal turbinates, pituitary gland, prostate gland, salivary glands, seminal vesicle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, parathyroid gland, tongue, trachea, uterus, Zymbals gland, and all lesions observed at autopsy. These tissues were fixed in 10% neutral buffered formalin, trimmed, processed in Innovative Medical System's LX300 tissue processor, infiltrated with paraplast, and embedded in Tissue Prep II. Sections were cut at 6/~m and stained with haematoxylin and eosin in a Shandon Veristain 24-3 automatic stainer. A cause of death or morbidity based on histopathological findings was given for all dead and moribund animals. Blood samples were taken at the 12- and 24-month killings for clinical chemistry evaluations. The following blood parameters were measured as a minimum: ?-glutamyl transpeptidase, total protein, glutamic-oxaloacetic transaminase, blood urea nitrogen, glutamic-pyruvic transaminase, creatinine, alkaline phosphatase, cholesterol, globulin, triglycerides, albumin, bilirubin and lactic dehydrogenase profile. Haematological evaluations were performed on samples from the same animals as described above. The parameters measured were: erythrocyte count, erythrocyte morphology, packed cell volume, haemoglobin, and leucocyte count (total and differential). Thyroid hormone assays consisted of analysis of serum samples for thyroid-stimulating hormone (TSH), total thyroxine (T4), total triiodothyronine (T3) and T3 uptake (Fullerton et al., 1987). Organ weights. The brain, liver, heart, thyroid gland, kidneys, testicles and ovaries from animals killed at 12, 18 and 24 months were weighed. Statistical procedures. The statistical procedure (CHRONIC) used in the analysis of tumour data is described by Kodell et al. (1983). CHRONIC follows a unified approach for the estimation and testing of the time-to-onset, prevalence, and mortality distribution functions. The onset and mortality functions represent 'net' rather than 'crude' probabilities in that they are adjusted for the mortality from causes of death other than the tumour of interest. The prevalence function of non-fatal tumours represents a probability further adjusted for mortality caused by the tumour. Specifically, the mortality function characterizes the mortality rate due to a tumour, the prevalence function characterizes the incidental (nonfatal) tumour rate, and the time-to-onset function characterizes the distribution of time to histological appearance of the tumour. Tests for overall doserelated trends in tumour incidence, along with onesided comparisons of each dose group with controls, were conducted. In reporting statistical significance for tumour incidence, the Bonferroni correction was applied to the 0.05 significance level to adjust the false positive error rate for multiple comparisons of several dose groups with the controls. A two-way mixed model analysis of variance was used to analyse the clinical chemistry data. Dose was considered to be a fixed variable while date and the dose/date interaction were considered to be random (Gaylor et al., 1987). The analysis involved a two-step method that first estimated the variance components of the model by the method of moments based on the expected

159

mean square for each component. Weighted least squares estimates of the means for each dose and Bonferroni confidence intervals for the difference of means of the dosed groups from the controls were calculated. To analyse organ weights and these weights as a percentage of terminal body weight, the dependent variables were analysed using a one-way Multivariate Analysis of Variance (MANOVA) with dose as the independent variable of interest. The organ weights or the ratios form a vector for each animal which was then analysed to determine whether there was a significant dose effect. The MANOVA design was used to incorporate the correlations of the organ weights within each animal, which were not included if separate ANOVAs were run for each organ. If the dose effect on the vector was significant, then six contrasts for each organ or ratio were examined using Bonferroni t-tests for significance. These contrasts were a test for linear trend and the five comparisons of the control group with each of the dosed groups. RESULTS

Body weights The average body weights for females and males are presented in Figs 1 and 2. The means were calculated for weeks 1-4 and then at 4-wk intervals for the remainder of the study. The body weight of the rats of both sexes in all groups increased rapidly at the same rate for approximately 20 wk, after which time the rate of gain of the animals in the high-dose group decreased and body weights from this time to the end of the study were less than those of the other groups. From approximately 65 wk until the end of the study, the average body weight of the control group was greater than any of the test groups. Feed consumption and dose rate Feed consumption followed a generally similar pattern in both sexes throughout the 2 yr. While the control group consumed slightly more feed than the test animals, this difference disappeared when the average feed consumption was calculated as g feed/g average body weight and was probably due to the slightly larger body weights of the control group. The average doses of SMZ consumed by the rats of the three higher dose groups plateaued after the rapid growth phase of the initial 20 wk, then decreased slowly throughout the remainder of the study. The dosage in the group receiving 2400 ppm in the diet decreased gradually from approximately 900mg SMZ/kg body weight/wk after 20 wk to about 650 mg for females and about 550 to 600 mg for males by the end of the study. The intake for the 1200 ppm group decreased gradually from about 450mg SMZ/kg body weight/wk during this same period, and the 600 ppm dose group ranged from approximately 200 to 150 mg of SMZ/kg body weight/wk. Mortality Mortalities from all causes excluding the animals killed are presented in Figs 3 and 4. An inverse dose-relationship was noted. After 2 yr on the study, the mortality of the females was approximately 41%

160

N.A. L1TTLEFIELDet al. 400

300 e 200

100 ,

O" o

.

.

lO

2o

.

.3 o .

.o

.

5o

60

;o

go

9'o

1vo

I,o

Experiment (weeks)

Fig. 1. Average body weights of female rats fed SMZ in the diet for 104 wk at dose levels (ppm) of 0 ( 10 (. . . . ), 40 ( - - - ) , 600 ( - - - - - ) , 1200 ( - - - - ) and 2400 (--).

,

.4oo]

!3°°t .~ 2 o o

0

.

.

10

.

.

20

30

.

40

6'0

50

7'0

8'0

;o

1 0' 0

Experi merrt (weeks)

Fig. 2. Average body weights of male rats fed SMZ in the diet for 104 wk at dose levels (ppm) of 0 ( 10 (. . . . ), 40 ( - - - ) , 600 ( - - - - - ) , 1200 ( - - - - ) and 2400 (- -), in the controls, compared with 36, 35, 26, 19 and 19% for the 10, 40, 600, 1200 and 2400 ppm dose groups, respectively. A m o n g the males, the mortality after 0.5.

,

2 y r on the study was approximately 37% in the controls and ranged from 24 to 28% in the treated groups.

Do~ 0000 .....

0010

------ 0040

0.4 '

----- 0600 ----1200 ---2400

0.3'

-40ppm

_TJ-"

{}

"2

,.j- 600 ppm

F

-" 1200ppm ,J~, ~ 2400 ppm

~"',~".~--- ~

0.2'

i~,/( 0.1

r I

o

1oo

|

200

!

300

Experiment

i ' 400

! 500

i 600

700

(days)

Fig. 3. Mortality rates of female rats fed SMZ in the diet for 104wk at dose levels of 0, 10, 40, 600, 1200 and 2400 ppm.

Carcinogenicity of sulphamethazine

161

0.5 Dose

.... D O 0 0 .....

0.4

00'10

0040 ----- 0600 ~-1200 ~2400 - - - - - -

O. 5 ~>'

~

/ /,.~

Control Dosed groups

0.'2

0.1

O

1; 0

2 0. 0

. 300 .

.4 0 0

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500

7 0' 0

E x p e r i m e n t (days)

Fig. 4. M o r t a l i t y . r a t e s o f m a l e r a t s fed S M Z in the diet f o r 104 w k a t d o s e levels o f 0, 10, 40, 600, 1200

and 2400 ppm.

Pathology

males (one each from the 1200 and 2400ppm dose groups) killed at 12 months of age in which a follicular cell adenoma of the thyroid gland was detected. The incidences of follicular cell adenocarcinoma of the thyroid glands for the groups killed after 18 and 24 months are presented in Table 2. In the 24-month group, the females had incidences of the latter tumours of 0.5, 0, 0, 0, 7 and 7% in the control, 10, 40, 600, 1200 and 2400 ppm dose groups, respectively. For males, the corresponding incidences were 0, 2, 0, 2, 2 and 8%. For combined thyroid gland follicular adenomas and adenocarcinomas, the incidences at 24 months were 4, 0, 1, 5, 10 and 9% in females and 0, 2, 0, 5, 5 and 11% in males of the control, 10, 40, 600, 1200 and 2400 ppm dose groups, respectively. The results of the statistical analysis (Table 3) indicated an overall trend towards increasing prevalence of follicular cell adenocarcinomas with increasing dose in both males and females. The differences for incidence of the thyroid neoplasia were significant in the female rats between the controls and the 1200 ppm group and for the male rats between the

A total of 1800 rats was allocated to the study, of which 1793 were autopsied and examined microscopically. Of the seven rats for which tissues were not examined, two were severely autolysed, three were lost in a laboratory accident, one was sent to the wrong destination (microbiology), and the tissues of one rat were lost. A total of 196 females and 187 males were removed from the study either dead or moribund. The cause of death of most of these animals was either mononuclear cell leukaemia or pituitary adenoma, the incidences of which were neither compound- nor treatment-related, although there was some evidence of an inverse dose relationship for mononuclear cell ieukaemia. The remaining animals died from various causes unrelated to the SMZ treatment. A dose-related neoplastic response was confined to the thyroid gland. The only thyroid follicular cell lesions noted, other than in the 18-month and 24-month dose groups, were in one male from the 2400 ppm group killed at 3 months of age, and in two

Table 2. Histopathological summary of thyroid follicular cell neoplasms in Fischer 344/N rats fed SMZ in the diet for 18 or 24 months Incidencet according to dose (ppm) after 18 months Type of thyroid neoplasm

0

10

Follicular cell adenocarcinoma

.

.

Follicular cell adenoma

--

--

Follicular cell adenoma or adenocarcinoma

--

--

Follicular cell adenocarcinoma

.

40 .

24 months

600

.

.

1/14 (7) 1/14 (7)

1200

2400

Females 1/170 (0.5) 1/15 5/170 (7) (3) 1/15 6/170 (7) (4)

. --

--

--

--

0

10

40

600

1200

2400

--

--

--

--

1/85 (l) 1/85 (1)

4/84 (5) 4/84 (5)

6/87 (7) 3/87 (3) 9/87 (10)

6/88 (7) 2/88 (2) 8/88 (9)

--

2/88 (2) 2/88 (2) 4/88 (5)

2/83 (2) 2/83 (2) 4/83 (5)

7/87 (8) 3/87 (3) 10/87 (1 !)

--

Males

Follicular

cell adenoma

Follicular cell adenoma or adenocarcinoma

2/15

(13) 2/15 (13)

.

. --

--

.

.

--

1/15

--

(7) 1/15 (7)

.

.

2/87 (2)

--

2/14

--

--

04) 2/14 (14)

--

--

2/87 (2)

--

--

*Dead and moribund animals are included as well as those killed. ?Incidence is expressed as the number of animals with the specified neoplasm/number of animals examined. Values in parentheses are the percentages of animals affected. A dash indicates that no neoplasms were observed among the animals of that group.

N. A. LITTLEFIELD

162

Table 3. Levels of significance (P-values) of positive trends among dose groups for neoplastic lesions in the thyroid gland of Fischer 344/N rats fed SMZ in the diet for 3, 12, 18 or 24 months Significance levels (P-values) Trends/groups considered

Females

Males

Follicular cell adenocareinomas

Overall Control Control Control Control Control

0.002** 0.709 0.712 0.797 0.005t 0.016

v. 10 ppm v. 40 ppm v. 600 ppm v. 1200 ppm v. 2400 ppm

0.00075*** 0.023 -0.043 0.045 0.008t

Follicular cell adenomas

Overall Control Control Control Control Control

0.355 0.944 0.611 0.294 0.532 0.492

v. 10 ppm v. 40 ppm v. 600 ppm v. 1200ppm v. 2400 ppm

0.00034*** 0.856 0.853 0.198 0.184 O.O07t

Follicular cell adenomas or adenocarcinomas

Overall Control Control Control Control Control

0.011 0.959 0.695 0.393 0.033 0.038

v. 10 ppm v. 40 ppm v. 600 ppm v. 1200 ppm v. 2400 ppm

0.00005*** 0.279 0.853 0.052 0.047 0.00008tit

**Significant trend at the 1.0% level. ***Significant trend at the 0.1% level. tSignificant trend at Bonferroni corrected 0.05 level (0.05/5). tttSignificant trend at Bonferroni corrected 0.001 level (0.001/5). c o n t r o l s a n d the 2400 p p m g r o u p . A significant difference w a s f o u n d for t h e 1200 p p m g r o u p a n d n o t for t h e 2400 p p m g r o u p o f female rats, d e s p i t e t h e s a m e incidence in t h e 2400 p p m animals. This r e s u l t e d b e c a u s e time was a f a c t o r in the statistical analysis, t h a t is, t h e t i m e - t o - t u m o u r was s h o r t e r for the neop l a s m s f o u n d in t h e rats o f t h e 1200 p p m g r o u p . I n

et

al.

m a l e rats, a n overall t r e n d t o w a r d s increased incid e n c e o f follicular cell a d e n o m a s o f t h e t h y r o i d g l a n d with increasing d o s e w a s f o u n d a n d the difference b e t w e e n the c o n t r o l s a n d t h e 2400 p p m d o s e g r o u p s was statistically significant. W h e n t h e a d e n o m a s a n d a d e n o c a r c i n o m a s were a d d e d , t h e differences b e t w e e n t h e incidence for the c o n t r o l a n d 1 2 0 0 p p m d o s e g r o u p s d i s a p p e a r e d for the females; t h e males cont i n u e d to exhibit a difference in the overall t r e n d a n d in the h i g h - d o s e g r o u p c o m p a r e d with t h e controls. Tests for differences in m o r t a l i t y , d u e to t h e lesions, were n o t d o n e since t h e r e w a s only o n e d e a t h t h a t was a t t r i b u t e d to a follicular cell n e o p l a s m o f the t h y r o i d gland. Follicular cell n e o p l a s m s were d i a g n o s e d as either cystic papillary a d e n o m a s o r a d e n o c a r c i n o m a s . T h e r e were 58 o f these n e o p l a s m s , 30 in females a n d 28 in males, w h i c h i n c l u d e d two o b s e r v e d at 12 m o n t h s (one m a l e rat in e a c h o f t h e 1200 a n d 2400 p p m g r o u p s ) a n d o n e m a l e fed 2400 p p m S M Z a n d killed at 3 m o n t h s . E i g h t o f the 58 n e o p l a s m s were in c o n t r o l animals. T h e d i s t r i b u t i o n w a s nearly equal b e t w e e n t r e a t e d m a l e a n d female rats as 24 were f o u n d in female rats a n d 26 were f o u n d in m a l e rats. M o s t o f these n e o p l a s m s (37 or 6 3 . 8 % ) were f o u n d in the t w o h i g h e s t d o s e g r o u p s . A d e n o c a r c i n o m a s (26) were f o u n d only in t h e rats killed at 24 m o n t h s a n d were d i v i d e d equally b e t w e e n m a l e a n d female rats. O f the 58 n e o p l a s m s , 32 were b e n i g n a n d 26 were c o n s i d e r e d to be m a l i g n a n t . T h e a d e n o c a r c i n o m a s were larger n e o p l a s m s a n d 22 o f the 26 were o b s e r v e d at a u t o p s y . O n l y nine o f the 32 b e n i g n n e o p l a s m s were f o u n d at a u t o p s y . Sixteen o f the a d e n o c a r c i n o m a s were invasive; the o t h e r s c o n t a i n e d areas o f a n a p l a s i a similar to t h a t o b s e r v e d in t h e invasive Table 4. Histopathological summary of non-neoplastic lesions lncidencet according to

90 days Type of lesion

0

10

40

600

12 months 1200

2400

0

10

40

600

1200

--

--

--

--

2/15 (13) 1/15 (7)

2400

Females

Thyroid gland, follicular cell - hyperplasia Thyroid gland, follicular cell, - focal cellular change Thyroid gland, follicle . cyst multilocular Eye, retinal atrophy .

--

--

.

.

.

.

.

Pancreas, acinus atrophy

.

.

.

.

.

.

Uterus, lumen dilatation

.

.

.

.

.

.

.

.

.

9/15 (60) .

. .

.

10/15 15/15 -(67) (100) 2/15 -(13) . . . .

.

Thyroid gland, follicular ceil - hyperplasia Thyroid gland, follicular cell, - focal cellular change Thyroid gland, follicle cyst, _ multilocular Eye, retinal atrophy .

.

.

.

.

.

Pancreas, acinus atrophy

.

.

.

.

.

.

L/t5 2]15 (7) (13) . . . .

.

5/15 (33) 6/15 (40) 1/15 (7)

14/15 15/15 (93) (100) . .

.

.

Males 15/15 1/15 (100) (7) 2/15 -(13) . . . 1/15 (7) 2/15 (13)

4/15 (27) 2/15 (13) 2/15 (13)

3/15 (20) 1/15 (7) 3/15 (20)

10/15 14/14 (67) (1001 2/15 1/14 (13) (7) 1114 (7) 5/15 6/14 (33) (43) 3/15 5/15 (20) (33) 1/15 -(7)

3/15 (20) 1/15 (7) .

1/15 (7) --

14/15 (93) 3/15 (20)

7/15 (47) 1/15 (7)

3/15 (20) 6/15 (40)

3/15 (20) 3/15 (20)

5/15 (33) 5/15 (33)

3/15 (20) 8/15 (53)

. 3/15 (20) 3/15 (20) 2/15 (13)

.

.

15/15 (100) 3/15 (20) 2]15 (13) 4/15 (27) 8/15 (53)

*Dead and moribund animals are included as well as those killed. tlncidence is expressed as the number of animals with the specified lesion/number of animals examined. Values in parentheses are the percentages of animals affected. A dash indicates that no lesions were observed among the animals of that group.

Carcinogenicity of sulphamethazine neoplasms. Several of the malignant neoplasms had very prominent areas of sclerosis. Invasion by the malignant tumours was limited to the tissues and organs adjacent to the thyroid. Many adenocarcinomas appeared to have developed directly from malignant changes in cystic papillary adenomas or multilocular cysts, but others were very hypercellular and their developmental origins were not apparent. The non-neoplastic lesions are presented in Table 4. The results of the statistical analysis for the non-neoplastic lesions (Table 5) indicated that dosing with S M Z resulted in dose-related lesions in the thyroid gland of both male and female rats. Overall trends and differences from controls were noted for treated animals for follicular cell hyperplasia, follicle cyst (multilocular), and focal cellular change of the follicular cell. Although there was a high incidence in the control animals, a dose response was noted for retinal atrophy. An overall dose-related trend occurred for acinus atrophy of the pancreas for both sexes but the incidence of this lesion was significantly different from the controls only for males at the two high doses. The analysis for dilation of the lumen of the uterus indicated a significant difference from controls for the 10, 40 and 2400ppm treatment groups, but no overall trend. Follicular cell hyperplasia was characterized by papillary infoldings of the follicular epithelium into the follicles. This resulted in irregularly shaped follicles, an obvious distortion of the normally round to oval follicles that are characteristic of the thyroid. Neither a pronounced increase in the size of the follicular cells nor multiple layers of these cells were features of this lesion, which was apparent only in follicles that contained little or no colloid. Frequently, a prominent capillary was present subjacent

163

to the basement membrane beneath the papillary projections. Hyperplasia was not usually diffuse except in those cases in which the severity was graded moderate. In the other cases, this lesion was focal to multifocal in its distribution, and usually the appearance was similar in both lateral lobes. Two or three follicles with prominent papillary projections into the follicular lumen were sufficient to diagnose minimal hyperplasia. A lesion of this type was seldom observed in control animals. The alterations of the follicles located at the periphery of the gland were not evaluated as critically as the internal follicles because they normally exhibit a wide variation in their histomorphology. The relationship to treatment for both sexes for all of the sacrifice periods was apparent only at doses of 600 ppm and more. Focal cell change of the follicular cell was a lesion characterized by focal aggregates of follicular epithelial cells whose cytoplasm was distended with a homogeneous eosinophilic colloidal-like material. In some instances these cells were arranged in a follicular pattern. Seldom did these aggregates consist of more than 50 cells. This lesion, like the follicular cell hyperplasia, was observed in the three highest dose groups of both sexes. This lesion was most prevalent in the animals killed at 18 and 24 months. The severity of the lesion remained constant in all dose groups and at all killing times. Multilocular cysts were also confined primarily to the three highest dose groups and were equally distributed between males and females. Nine of these cysts were observed at autopsy and eight of these were in animals in the 2400 ppm, 24-month group. Morphologically, this lesion was characterized by a single cyst divided into multiple compartments. The morphology of the epithelial lining of these cystic

in Fischer 344/N rats* fed SMZ in the diet for 3, 12, 18 or 24 months dose (ppm) after 24 months

18 months 0

10

40

600

1200

2400

0

10

40

600

1200

2400

3/170 (2) 3/170 (2) 3/170 (2) 74/175 (42) 71/179 (40) 6/179 (3)

1/86 (I) 3/86 (3) --

3/85 (4) 1/85 (1) --

38/88 (43) 25/90 (28) 9/90 (10)

48/88 (55) 30/90 (33) 7/90 (8)

49/84 (58) 5/84 (6) 3/84 (4) 43/86 (50) 37/87 (43) 4/89 (4)

66/87 (76) 12/87 (14) 7/87 (8) 63/85 (74) 35/86 (41) 11/89 (12)

86/88 (97) 21/88 (24) 15/88 (17) 81/90 (90) 47/90 (52) 13/90 (14)

9/170 (5) 9/170 (5) 3/170 (2) 60/173 (35) 107/176 (61)

3/87 (3) 4/87 (5) 2/87 (2) 30/84 (36) 49/88 (56)

7/84 (8) 1/84 (1) 1/84 (l) 30/87 (34) 58/89 (65)

39/88 (44) 5/88 (6) 5/88 (6) 36/88 (41) 52/89 (58)

63/83 (76) 11/83 (13) 7/83 (8) 57/87 (66) 64/88 (73)

82/87 (94) 16/87 (18) 16/87 (18) 66/87 (76) 69/89 (78)

Females

--

--

--

--

--

--

.

.

.

.

11/15 (73) 1/15 (7)

6/15 (40) --

.

3/15 (20) 5/15 (33) --

6/15 (40) 3/15 (20) 2/15 (13)

10/15 (67) 4/15 (27) 4/15 (27)

5/15 (33) 7/15 (47) 1/15 (7)

6/15 (40) 9/15 (60) --

2/15 (13)

--

1/14 (7) 1/14 (7) 1/14 (7) 4/14 (29) 3/15 (20)

5/15 (33) 2/15 (13) --

8/14 (57) 3/14 (21) 1/14 (7) 7/14 (50) 10/15 (67)

14/15 (93) 2/15 (13) 1/15 (7) 14/15 (93) 7/15 (47) -Males

5/15 (33) 11/15 (73)

1/14 (7) -5/14 (36) 7/14 (50)

7/15 (47) 9/15 (60)

13/14 (93) 3/14 (21) 4/14 (29) 5/14 (36) 9/14 (64)

N. A. LITTLEFIELD et al.

164

Table 5. Levels of significance(P-values) for positive trends among Fischer 344/N rats fed SMZ in the diet for Significance (P value) Lesion and comparison Thyroid gland, follicular cell hyperplasia Overall Control v. 10 ppm Control v. 40 ppm Control v. 600 ppm Control v. 1200ppm Control v. 2400ppm

Females

Males

0.000051"I"I" 0.504 0.103 0.00005*** 0.00005*** 0.00005***

0.000051"1"t" 0.513 0.115 0.00005*** 0.00005*** 0.00005***

Thyroid gland, follicular cell, focal cellular change Overall Control v. 10 ppm Control v. 40 ppm Control v. 600 ppm Control v. 1200ppm Control v. 2400 ppm

0.00005ttt 0.075 0.341 0.037 0.0006** 0.00005***

0.00005tt~" 0.203 0.786 0.134 0.017 0.00006***

Thyroid gland, follicle cyst, multilocular Overall Control v. 10 ppm Control v. 40 ppm Control v. 600 ppm Control v. 1200ppm Control v. 2400ppm

0.00005ttt 0.877 0.873 0.325 0.069 0.00005***

0.00005ttt 0.338 0.313 0.067 0.027 0.00005***

Eye, retinal atrophy Overall Control v. 10 ppm Control v. 40 ppm Control v. 600 ppm Control v. 1200ppm Control v. 2400ppm

0.000051"1"I" 0.267 0.007* 0.495 0.0002** 0.00005***

0.000051"?? 0.284 0..264 0.074 0.0004** 0.00005***

Pancreas, acinus atrophy Overall Control v. 10 ppm Control v. 40 ppm Control v. 600 ppm Control v. 1200ppm Control v. 2400ppm

0.003tt 0.983 0.907 0.511 0.466 0.030

0.00005I"t"I" 0.680 0.661 0.670 0.008* 0.002*

Uterus, lumen dilatation Overall 0.187 -Control v. 10 ppm 0.002* -Control v. 40 ppm 0.002* -Control v. 600 ppm 0.098 -Control v. 1200ppm 0.015 -Control v. 240.0ppm 0.006* -ttSignificant trend at the 1.0% level. tttSignificant trend at the 0.1% level. *Significant trend at Bonferroni corrected 0.05 level (0.05/5), **Significant trend at Bonferroni corrected 0.01 level (0.01/5). ***Significant trend at Bonferroni corrected 0.001 level (0.001/5). spaces was variable. T h e epithelium was sometimes flattened, a n d on occasion the follicular lining cells were very p r o m i n e n t a n d occasionally formed a few papillary projections. All o f these patterns were sometimes observed in a single multiloculated cyst. The cysts were usually distended with colloid. There was never m o r e t h a n one multiloculated cyst diagnosed in a single thyroid. A relationship of multilocular cysts to cystic papillary a d e n o m a s a n d a d e n o c a r c i n o m a s was apparent. A c o n t i n u u m f r o m multilocular cysts to cystic papillary a d e n o m a s or a d e n o c a r c i n o m a s a p p e a r e d in several cases. Diagnostic difficulties occurred in a few cases in which there were histopathological changes t h a t could be interpreted as representing either a multilocular cyst or a neoplasm.

T h e ocular lesion o f the earliest, m o s t minimal retinal atrophic change was a decrease in thickness (loss of cells) in the outer nuclear a n d the rod a n d cone layers. As the severity of the a t r o p h y increased, o t h e r layers were either absent (total a t r o p h y ) or reduced in thickness. In m a n y o f the m o r e severely affected retinas there was a p r o n o u n c e d retinal neovascularization a n d vascular sclerosis. The combin a t i o n o f these lesions resulted in a retina t h a t was severely disorganized, a n d in some cases only r e m n a n t s o f the retina were recognizable. A severe form o f endstage ocular degeneration (phthisis bulbi) was diagnosed in 65 rats, a n d it occurred almost exclusively in the animals killed at 24 m o n t h s . Males a n d females were approximately equally affected. The severest form o f this lesion was sometimes recognized at autopsy a n d the affected eye was described as white, opaque, a n d / o r small. Histologically, this severe endstage degenerative condition was characterized by a severe lenticular degeneration (cataract), a n d retinal a t r o p h y (degeneration). In addition, o t h e r a c c o m p a n y i n g lesions frequently present were: loss o f vitreous h u m o u r , collapse o f the vitreal cavity, a d h e s i o n o f the lens to the retina, vascular proliferation, distention of the anterior c h a m b e r a n d posterior synechia. Cataracts (lenticular degeneration) were observed frequently in rats o f b o t h sexes, t h a t is 189 females a n d 178 males. This lesion was observed only in the lenses of rats t h a t also h a d retinal atrophy.

Clinical chemistry Dose-response trends t h a t were observed f r o m the analysis o f results for cholesterol, lymphocytes, a n d h a e m o g l o b i n did n o t occur consistently at the different killing times. In males at 24 m o n t h s , cholesterol levels were lower t h a n those of the controls a n d lymphocyte levels were elevated for the 2400 p p m t r e a t m e n t group. In females at 12 m o n t h s , h a e m o globin c o n c e n t r a t i o n s were low in the treated groups c o m p a r e d with the control groups. N o toxicological significance was attributed to these results due to the apparently r a n d o m variations.

Organ weights The weights recorded for the thyroid gland, liver, h e a r t a n d kidneys at autopsy are s u m m a r i z e d in Table 6. There were n o significant dose-related weight changes in the brain, testicles or ovaries. T h y r o i d gland weight showed highly significant linear trends in each group a n d b o t h sexes, with thyroid weight increasing at the higher doses o f SMZ, a n d highly significant differences between controls a n d the 2400p p m dose groups, a n d also between the 1200-ppm group o f females at 12 a n d 24 m o n t h s . The analysis o f the weights for the liver, heart a n d kidneys showed linear trends for some groups, with weights t h a t were significantly lower for the treated rats. While weight differences for the h e a r t occurred for females, the males d e m o n s t r a t e d n o changes related to treatment. T h e kidney weights were different f r o m the controls for the top three t r e a t m e n t groups for b o t h sexes at the 2 4 - m o n t h kill. The significant differences in the o r g a n : b o d y weight ratios c o r r e s p o n d e d reasonably well with the organ-weight d a t a described above, with one notable exception. While analysis o f the b r a i n

Carcinogenicity of sulphamethazine

165

Table 6. Organ weightsat autopsy for Fischer344/N rats fed SMZ in the

diet

for 12, 18 or 24 months

Organ weights according to dose (ppm) Organ

Time of killing

Sex

Linear trend

0

10

40

600

1200

2400

Weight(rag) Thyroid

12 18 24 12 18 24

F F F M M M

*** *** *** *** *** ***

15.3+3.1 24.1+2.4 23.5_+5.9 19.3_+2.1 30.6_.+9.6 29.2_+8.9

15.3+2.1 24.5+2.8 22.8_+5.0 19.4_+2.7 29.5+4.4 30.3_+8.0

15.7_+2.3 27.5+12.3 22.7_+11.4 20.1 + 3 . 3 27.1 _+3.3 27.7_+7.1

15.3+2.0 24,8-+3.2 26,1_+10.4 20.6_+3.1 29.0-+4.8 29.8_+6.8

18.7+2.5" 30.2-+4.0 28.8_+7.3** 22.4+3.4 37.5-+ 11.4 32.1_+6.3

24.2+2.3*** 36.5_+2.8*** 38.9_+9.3*** 34.2-+3.7*** 47.5_+7.2*** 45.3_+11.4"**

Liver

12 18 24 12 18 24

F F F M M M

** ----***

6.1_+1.1 7.7_+0.5 9.6-+1.8 11.1+_2.9 12.8_+0.9 13.2_+ 1.7

6.7_+0.04 8.0_+0.6 8.6-+1.1" 12.5+_1.3 12.4_+1.5 12.8_+ 1.8

6.8_+0.4 7.8_+0.7 8.9_+1.3 12.5_+1.4 11.7_+0.8 12.8_+3.2

6.4_+0.3 7.7_+0.7 9,0+1.8 12,3_+1.2 11.9_+1.1 12.1 _+ 2.1"

6.2_+0.3 7.5-+0.7 8.9_+2.1 12.2_+1.2 12.1_+0.8 11.7_+ 1.7"**

6.1+0.3 7.3-+0.4 8.6_+1.1"* 11.9_+0.7 12.5_+1.1 11.0_+ 1.3"**

Heart

12 18 24 12 18 24

F F F M M M

-* -----

0.80 _+ 0.09 0.89 _+ 0.05 0.98 _+ 0.11 1.40_+0.18 1.31 _+0.08 1.37-+0.17

0.80 _+0.09 0.87 _+ 0.08 0.92 _+ 0.09* 1.40_+0.17 1.32_+0.17 1.40_+0.25

0.80 _+ 0.06 0.87 _+ 0.06 0.92 _+ 0.09* 1.35_+0.19 1.32_+0.20 1.30_+0.17

0.78 _+ 0.08 0.87 _+0.06 0.92 _+ 0.12" 1.35_+0.18 1.20_+0.06 1.35-+0.26

0.77 _+ 0.04 0.84 _+ 0.07 0.92 _+0.08** 1.23_+0.15 1.31 + 0 . 1 4 1.30_+0.19

0.76 _+0.08 0.80 _+ 0.06 0.91 4- 0.10"** 1.27_+0.15 1.30-+0.12 1.28-+0.17

Kidney't

12 18 24 12 18 24

F F F M M M

*** ** *** ** -***

0.82 _+ 0.04 0.98 _+ 0.05 1.08 _+ 0.08 1.36_+0.10 1.49 _+ 0.07 1.59_+0.14

0.85 _+ 0.05 0.96 _+ 0.07 1.02 _4-0.08** 1.44_+0.09 1.52 _+ 0.15 1.57_+0.17

0.85 _+ 0.05 0.97 _+ 0.05 1.04 -+ 0.07 1.41 _+0.12 1.44 +- 0.05 1.53_+0.18

0.81 _+ 0.05 0.94 _+ 0.06 1.01 _+0.07*** 1.34_+0.13 1.44 _+ 0.13 1.50_+0.14"*

0.75 _+ 0.12 0.94 _+ 0.04 0.99 -+ 0.07*** 1.34_+0.11 1.47 _+0.07 1.45_+0.12"**

0.75 +_0.05 0.89 _+ 0.04* 1.00 _+ 0.08*** 1.27_+0.07 1.47 _+ 0.09 1.44_+0.14"**

Weight(g)

tRight kidneyweight. Values are means+_SD; those marked with asterisks show significantdifferencescompared with the correspondingcontrol value significanttrends) (*P < 0.05; **P < 0.01; ***P < 0.1). weights by themselves revealed no treatment-related trends or differences, the brain:body weight ratio at 24 months was significantly different from controls in both males and females and at all dose levels. DISCUSSION

In a 90-day study, SMZ produced follicular cell hyperplasia of the thyroid gland in treated rats but this lesion was not observed in treated mice. Therefore, our chronic studies on SMZ were designed with a high dose of 4800 ppm for the mice (Littlefield et al., 1989) and 2400 ppm for the rats. The mice killed after 24 months had follicular cell adenomas of the thyroid gland (Littlefield et al., 1989) while as shown in this report, the treatment-related neoplastic lesions of the thyroid gland in the rats were follicular cell adenocarcinomas. Apart from the thyroid lesions, there were no other treatment-related neoplastic lesions from SMZ treatment in either the rat or mouse. In addition, the treatment-related lesions did not appear until late in the lifespan of the animals. While there were some follicular cell adenomas of the thyroid gland in the rats, a dose-related difference from controls occurred only in the males of the high-dose group. Morphologically, there appeared to be a direct relationship between the adenomas and the adenocarcinomas. The development of non-neoplastic lesions provided further substantiation that the thyroid gland was a target organ for SMZ toxicity. A very high incidence of hyperplasia of the thyroid follicular cells was noted after only 90 days in the rats from the three highest dose groups. The severity of hyperplasia was greatest in the rats killed after 3 months. Thyroid

(or

follicles in untreated 3-month-old rats appear to be morphologically far more active than those in the 18and 24-month-old control animals. The hyperplasia may be induced more readily in the active glands of the younger animals, and the older animals are probably more refractory. The stages of follicular cell oncogenesis in laboratory animals have been demonstrated by Boorman (1983) and Frith and Heath (1983) to be: (1) diffuse hyperplasia, which lasts until about the eighth month of treatment, (2) nodular proliferation of the follicular cells with formation of benign tumours, lasting up to about 18 months, (3) malignant tumour growth, beginning after about 18 months of treatment. Sulphonamides are known to induce thyroid dysfunction in rats (MacKenzie and MacKenzie, 1943) and inhibit thyroid hormone synthesis (Ganong, 1974). Toxicological/carcinogenic effects on the thyroid gland, as demonstrated in this study, substantiate the goitregenic effects of the sulphonamide, SMZ. Bridges et al. (1968) reported that there were demonstrable differences between humans and rats in the metabolism of another sulphonamide, sulphadimethoxine. Whether this difference would result in either increased or decreased risk to humans is speculative at this point. The analysis of the serum triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH) and T3 uptake, after oral administration of SMZ to rats, has been considered by Fullerton et al. (1987). Although the serum T3 levels were not significantly different between dose groups (P > 0.05) for either sex, there was a tendency for the levels to decrease with increasing dose levels and with age. Serum T4 levels were significantly lower (P < 0.05) for females

166

N. A. LITTLEFIELDet al.

in the 1200 and 2400-ppm dose groups at 18 months and for males at 600, 1200 and 2400ppm at 24 months than the respective levels in the control animals. The T4 levels declined with age for both males and females. Although not statistically significant, probably due to a large standard deviation and variance between the samples, the mean TSH serum levels were greater in both sexes in the 600, 1200 and 2400-ppm dose groups than in the control animals. The TSH levels also declined as the rats aged. There were no differences between the dose groups in the percentage uptake of T3. The results indicate that the treatment of rats with SMZ caused enlargement of the thyroid gland. This enlargement of the thyroid glands was probably a result of the sustained increase in TSH levels. Ganong (1974) has demonstrated that sulphonamides inhibit thyroid hormone synthesis. The reduced circulating thyroid hormones causes a rise in the release of TSH from the pituitary gland. A sustained production of TSH ultimately causes a continuous stimulation of the thyroid gland which eventually results in thyroid hyperplasia. Since serum T3 and T4 levels control pituitary output of TSH, a reduction of the thyroid hormone:pituitary hormone ratio indicates that the thyroid gland has a dysfunction, causing the pituitary to produce an excess amount of TSH. It is evident that thyroid hyperplasia is caused by increased and sustained release of TSH from the pituitary gland. Prolonged stimulation of the pituitary, as would occur in a chronic dosing study, may cause the hyperplasia to progress to neoplasia. Since hyperplasia often regresses upon removal of the stimulus, a threshold for thyroid follicular neoplasia may be indicated since some degree of inhibition of the thyroid gland can be accommodated within the bounds of the normal feedback mechanism. Todd (1986) showed that thyroid follicular cell adenomas that were caused by sulphonamides regressed upon cessation of exposure. Thyroid follicular cell adenocarcinomas occur spontaneously in the strain of rat used in this study. Haseman et al. (1984) presented data from untreated Fischer 344 rats from several chronic studies in which these rats had an average background incidence of thyroid follicular cell adenocarcinomas of slightly less than 1% (17/2230, 0.8%). If SMZ is augmenting an already existing carcinogenic process, low-dose linearity is expected (Crump et al., 1976). Thyroid follicular cell adenocarcinomas of both sexes in this study had a linear term in the best-fitting multistage model. The occurrence of a treatment-related effect on retinal atrophy was unexpected and difficult to explain. Retinal atrophy, or as it is commonly referred to in the literature, retinal degeneration, is a common phototoxic ocular lesion in albino rats maintained in a laboratory environment. This change is believed to occur as a result of exposure to fluorescent or incandescent light and also possibly due to ageing (Greenman et al., 1982). The lesion produced by these two causes was similar and cannot easily be differentiated by its histomorphological features. There is normally a detectable level of atrophic changes (thinning of all layers of the retina) in animals maintained on an experiment for 18-24 months. In this study, atrophy was diagnosed only in

those rats in which the severity was greater than that customarily present. Even with this diagnostic limitation, it was still apparent that treated males and females had a significant increase in retinal atrophy when compared with the incidence in the control group. This increase in incidence for the 24-month animals was apparent for all the female groups (except in the 10 ppm group), and in the males of the 1200 and 2400 ppm groups. Cataracts were present only in rats that also had retinal atrophy. The cause of the cataracts is not known. No reference that would implicate a phototoxic effect was found; however, the circumstantial evidence in this study is compelling. For instance, 21 of 31 rats with cataracts from the 18-month sacrifice period were caged on the top shelf. A similar pattern was also observed in the rats killed at 24 months, in that 190 of 268 with cataracts were also caged on the top shelf. The cataract degeneration may be secondary to that in the retina. The vascular lesions in the retinas may also have resulted in impairment of circulation to other ocular structures, including the lens. Transparency of the avascular lens can be adversely affected by inadequate ocular circulation. These data were also analysed for effects from other sources, such as the position of the respective columns on the rack. There is evidence as shown in the 24-month group that the position of the cage on the rack is not the only influencing factor. One possible explanation would be that SMZ may have a photosensitizing influence that makes the rats more susceptible to this lesion, thus accounting for the treatment effect. Definite conclusions cannot be drawn about the phototoxic effects of SMZ, without a further study designed to allow the accurate measurement and control of the total light exposure of each cage.

REFERENCES

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(1983) CHRONIC: An SAS procedure for statistical analysis of carcinogenesis studies. J. statist, comput. SimuL 16, 287-310. Littlefield N. A., Blackwell B.-N., Allen R. R. and Gaylor D. W. (1989) Chronic toxicity and carcinogenicity studies of sulfamethazine in B6C3Fj mice. Fd Chem. Toxic. 27, 455-463. MacKenzie C. G. and MacKenzie J. B. (1943) Effect of sulfonamides and thiourea on thyroid gland and basal metabolism. Endocrinology 32, 185-209. Pan S. W. (1948) Effect of sulfamethazine on the testis and accessory glands of normal and hypophysectomized rats. Proc. Soc. exp. Biol. Med. 68, 488. Swarm R. L., Roberts G. K. S., Levy A. C. and Hines L. R. (1973) Observations on the thyroid gland in rats following the administration of sulfamethoxazole and trimethoprim. Toxic. appl. Pharmac. 24, 351-363. Todd G. C. (1986) Induction and reversibility of thyroid proliferative changes in rats given an antithyroid compound. Vet. Pathol. 23, 110-117.