14,179-190(1990)

FlJNDAMENTALANDAPPLIEDTOXlCOLOCY

Chronic Toxicity and Carcinogenicity of Methylmercury Chloride in B6C3Fl Mice K. MITSUMORI, M. HIRANO, H. UEDA, K. MAITA, AND Y. SHIRASU Institute

ofEnvironmental

Received

Toxicology,

April

-T-772 Suzuki-cho.

IO, 1989; accepted

Kodaira-shi,

August

Tokyo

187, Japan

9, 1989

Chronic Toxicity and Carcinogenicity of Methylmercury Chloride in B6C3Fl Mice. MITSUMORI,K.,HIRANO,M.,UEDA,H..MAITA,K.,ANDSHIRASU,

Y.(1990).Fundam.Appl.

Toxicol.

14, 179- 190. A 2-year feeding study of methylmercury chloride (MMC: 0, 0.4, 2, or 10 ppm) was conducted in B6C3FI mice (60 mice of each sex/group) to compare chronic toxicity and carcinogenicity results with those for ICR mice from our previous study in which males of the IO-ppm group showed an increased incidence of renal tumors without any abnormal in-life parameters. In B6C3FI mice of the IO-ppm group, neurotoxic signs characterized by posterior paralysis were observed in 33 males after 59 weeks and in 3 females after 80 weeks. In males, a marked increase in mortality and a remarkable decrease in body weight gain were observed after 60 weeks. Toxic encephalopathy consisting of neuronal necrosis of the brain and toxic peripheral sensory neuropathy were induced in both sexes in this group. Chronic nephropathy, testicular atrophy, and glandular stomach ulcer increased in incidence in the males; chronic nephropathy also increased in incidence in females. In proliferative lesions, there were significant increases in the incidence of renal adenoma and/or carcinoma ( I6/60) and tubular cell hyperplasia ( 141 60) in males of the IO-ppm group, as compared to the control group. The incidence of chronic nephropathy also increased in males of the 2-ppm group. The results of this study indicate that the susceptibility of B6C3Fl mice to renal toxicity and renal carcinogenicity is comparable to that of ICR mice, and B6C3Fl mice are more sensitive to the chronic neurotoxic effectsof MMC than are ICR mice. o 1990 Society ofToxicology.

Various strains/hybrids of mice, such as ICR Swiss, BALB/C, C57BL, and B6C3F1, have been used in long-term toxicity studies to detect chronic toxic effects and carcinogenicity of chemical substances. Among these mice, the random-bred ICR Swiss albino mice have been widely used for a long time in long-term toxicity studies for the safety evaluation of chemicals by the chemical and pharmaceutical industries. On the other hand, the U.S. National Cancer Institute (NCI) decided in 1972 that the B6C3Fl mouse was the most appropriate for long-term studies using mice since other strains/hybrids, including ICR strain, had a shorter life span and/or a high incidence of lymphomas and vascular tumors (National Toxicology Program, 1984). Since then the NC1 and the National Toxicol-

ogy Program (NTP) have been using B6C3Fl hybrid as the standard mouse for chronic toxicity and carcinogenicity studies (Rao et al., 1988). Which strain/hybrid is the most sensitive model for toxicity testing has not been rigorously tested; there might be no essential difference in the onset of toxic effects of chemicals among the strains/hybrids of mice. We have conducted long-term studies in rats (Mitsumori et al., 1983, 1984) and mice (Mitsumori et al., 1981; Hirano et al., 1986) to examine chronic toxic effects and carcinogenicity of methylmercury chloride (MMC). Our studies using ICR mice demonstrated that renal epithelial tumors were induced only in male mice fed diets containing 15 or 10 ppm of MMC for 78 weeks or longer (Mitsumori et al., 1981; Hirano et al., 1986). In 179

0272-0590190 $3.00 Copyright 0 1990 by the Society of Toxicology. All rights of reproduction in any form reserved.

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80706050-

ET AL.

Male

t2100go-

= e = -----------m-v, * c * 5 = *

80-

Female

7060-

I 8

0

=

-___---___0.4 ppm

I I I I I 16 24 32 40 48

I

I

I

56 64 72

I

I

I

I



80 88 96 104 Weeks

FIG.

1. Survival curves for B6C3Fl mice fed diets containing MMC for 104 weeks.

addition, we obtained a result indicating that testosterone may have an important role in the induction of renal tumors in ICR mice by MMC (Hirano et al., 1988). These results were obtained in ICR mice, and it is not known whether B6C3F I mice are more sensitive to the carcinogenic and/or toxic effects of MMC than ICR mice. Therefore, we conducted a 2-year long-term MMC feeding study in B6C3F 1 mice, with the same dosages used in our previous study in ICR mice (Hirano et al., 1986), to compare the results of both studies with respect to chronic toxicity and carcinogenicity. MATERIALS

AND

METHODS

Methylmercury chloride, 99.3% purity, was obtained from Tokyo Kasei Kogyo Company. Ltd., Japan. Four-

week-old B6C3Fl specific-pathogen-free mice (260 males and 260 females) were purchased from Charles River Japan Inc., Atsugi, Kanagawa, Japan. Four animalsofeither sex were housed in an aluminum cage with a wire-mesh floor in a barrier-system animal room controlled at 24 + 1°C and 55 & 5% humidity. After a l-week acclimation period, mice of both sexes were divided into four groups, each consisting of 60 males and 60 females. MMC was mixed with a commercial powdered chow M (Oriental Yeast Ltd., Japan) at 0 (control), 0.4,2, and 10 ppm, and fed to the mice for 104 weeks. Animals were observed daily for their general condition. The body weight of each animal was recorded weekly during the first 26 weeks and biweekly thereafter. Food consumption was measured twice a week for four representative cages in each group. Animals found dead, animals killed in extremis during the study, and all animals surviving 104 weeks were autopsied. Tissues of all organs from all animals were fixed in 10% phosphatebuffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. Organs and tissues histopathologically examined were those listed on the section

METHYLMERCURY

I

z?

I

I

I

1

TOXICITY

I

I

IN B6C3Fl

1

I

181

MICE

I

I

I

I

I

mo

60-

0 PPm --_---- __- 0.4 ppm ------

-

0

I

8

I

16

I

24

I

32

I

40

I

48

I

56

I

64

Pm

2

10 ppm I I I

72

80

88

I

I

96

104

Weeks FIG.2. Group mean body weights of B6C3Fl mice fed diets containing MMC for 104 weeks.

on oncogenicity study in Pesticide Assessment Guidelines of U.S. Environmental Protection Agency (Jaeger, 1984); all gross lesions and masses were also examined. Fisher’s exact test was used to determine the statistical significance of the incidence of histopathological lesions between the control group and each ofthe treated groups.

RESULTS Neurological signs from posterior paresis to paralysis were first observed at Week 59 in males and at Week 80 in females of the loppm group. The number of mice showing these signs gradually increased in males of this group thereafter. In the lo-ppm group, these neurological signs occurred in 33 of 60 males and 3 of 60 females. A marked increase in mortality was observed for males of the loppm group after Week 60; this was accompanied by an increase in the severity of the neu-

rological signs. The final survival rate of males in this group was 17%, in contrast to 48% in the male controls (Fig. 1). Body weight gain in the IO-ppm group was remarkably inhibited after Week 60 in males and after Week 92 in females, but not in the other treated groups (Fig. 2). A significant decrease in food consumption was seen in males of the 10-ppm group after Week 56 as compared to the control group. The overall mean daily intakes of MMC in the lo-, 2-, and O.Cppm groups were 0.859,O. 174, and 0.0382 mg/kg/ day for males and 0.752, 0.166, and 0.0332 mg/kg/day for females, respectively. Macroscopically, there was an increased incidence of testicular atrophy and kidneys that were pale and granular in males of the IO-ppm group after Week 52. A renal tumor mass was first seen in a male from the IO-ppm

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ET AL.

TABLE 1 INCIDENCE

OF TREATMENT-RELATED NONNEOPLASTIC LESIONS OF TARGET IN B6C3Fl MICE TREATED WITH MMC

ORGANS

Number of mice with tumors Male

Female

Dose level (ppm): Number of mice examined:

0 60

0.4 60

2 60

10 60

0 60

0.4 60

2 60

10 60

PNS”: Sensory neuropathy Cerebrum: Neuronal necrosis Cerebellum: Neuronal necrosis Kidney: Chronic nephropathy Testis: Tubular atrophy Glandular stomach: Ulcer

0 0

0 0

0 0

45** 16**

0 0

0 0

0 0

4 2

0 8 1 1

0 12 5 1

0 27** 2 0

21** 59** 54** 7*

0 5 0

0 6 0

0 3 1

3 56** 0

’ Peripheral nervous system. * p < 0.05: **p < 0.01 (Fisher’s exact test), as compared to the control.

group killed in extremis at Week 70. A total of 10 male mice in the IO-ppm group had a renal mass. No renal masses were macroscop-

ically evident in females of this group or in animals of either sex in the other treated groups.

FIG. 3. Spinal ganglion (G), spinal dorsal roots (D), and ventral roots (V) from a control male B6C3Fl mouse. H&E, X20.

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183

FIG. 4. Severe degeneration of myelinated nerve fibers in the spinal ganglion (G) and dorsal roots (D) of the cauda equina from a male B6C3Fl mouse in the 10-ppm group with neurological signs. No remarkable change in the ventral roots(V). H&E, X20.

Histopathological nonneoplastic lesions that exhibited statistically significant increases in overall incidence are listed in Table 1. Peripheral sensory neuropathy occurred in 45 males and 4 females in the 10-ppm group. This neuropathy was characterized by degeneration of myelinated nerve fibers in the spinal dorsal nerve roots (Figs. 3, 4) and sciatic nerves and neuronal loss in the spinal ganglia. Similar degeneration of myelinated nerve fibers was also present in the spinal dorsal funiculus corresponding to the ascending pathway of the peripheral sensory nerves. In mice from the lo-ppm group that showed neurological signs, neuronal necrosis occurred in the cerebral cortex, caudate nucleus, and cerebellar granular layer (Figs. 5,6). In total, this neuronal necrosis in the cerebellum occurred in 27 of 60 males and 3 of 60 females. Chronic nephropathy, consisting of epithelial degeneration and regeneration of the

proximal tubules and interstitial fibrosis, significantly increased in both sexes in the loppm group as compared to the control group. The overall incidence was 59 of 60 males and 56 of 60 females, but the renal damage was generally more prominent in males than in females. Similar nephropathy was also observed in males of the 2-ppm group with a significantly higher incidence. This nephropathy was less severe in the 2-ppm group than in the 10-ppm male group. In males of the IO-ppm group, tubular atrophy of the testis was frequently observed and the incidence of ulcer in the glandular stomach significantly increased as compared to the control. Although various kinds of spontaneous nonneoplastic lesions other than the above lesions were seen in treated groups, the incidences of these lesions showed no statistically significant differences from that of the control group.

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FIG. 5. Cerebellum from a control male B6C3Fl mouse. H&E, X40.

Epithelial proliferative lesions of the kidney that showed statistically significant increases in overall incidence in treated groups are listed in Table 2. The overall incidence of renal epithelial tumors in males of the loppm group was 16 of 60, which represented a significant increase as compared to the control (O/60). Renal tumors were first observed microscopically at Week 60. Among the animals with these tumors, 13 were diagnosed as carcinomas and 5 as adenomas. These tumors were seen in the unilateral kidney of mice that died or were killed after Week 60. The renal carcinomas were well demarcated from normal tissue and classified as solid or cystic papillary type. The solid type showed a pseudoglandular or solid pattern composed of epithelial cells with nuclear pleomorphism (Fig. 7). The papillary type was characterized by extensive papillary growth of cuboidal to polyhedral cells with bizarre nuclei into large cystic spaces. No metastases were seen in the animals with renal carcinomas. Adenomas

found in the IO-ppm group were classified as solid or tubular type. The solid type was a solitary small nodule consisting of closely packed polygonal cells and was well demarcated from compressed adjacent renal parenchyma. The tumor cells had eosinophilic granular cytoplasm or clear cytoplasm. There was no nuclear pleomorphism, and mitotic figures were rare. The tubular type consisted of multiple enlarged tubules lined by one to two layers of neoplastic epithelial cells that morphologically resembled the normal proximal tubular epithelium but contained more cytoplasm. Nuclear pleomorphism was not evident. Such an adenoma was also observed in a female of the IO-ppm group and a male of the 2-ppm group. There were no renal tumors in males treated with 0.4 ppm or in females treated with 2 ppm or less. Focal hyperplasia of the renal tubules was seen only in males of the 10-ppm group. The incidence was 14 of 60, and was statistically significantly different from the control. This

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FIG. 6. Neuronal necrosis of the cerebellar granular cell layer from a male B6C3Fl mouse in the IO-ppm group. H&E. X40.

hyperplasia was characterized by solid growth of tubular epithelial cells occluding the tubular lumen (Fig. 8) or simple papillary growth of atypic eosinophilic cells in dilated cystic tubules. Besides the renal proliferative lesions, no other tumors had a significant increase in incidence in the treated groups. DISCUSSION In our previous study using ICR mice (Hirano et al., 1986) in-life parameters showed no abnormal changes in mice of both sexes fed diets containing 10 ppm of MMC, as shown in Table 3. The overall mean daily intakes of MMC (mg/kg) in the IO-, 2-, and 0.4ppm groups in our previous study (Table 3) were comparable to those in the present study. However, male B6C3Fl mice of the lo-ppm group in the present study showed

frequent occurrence of neurological signs, a marked increase in mortality, and a remarkable decrease in body weight gain. In addition, similar neurological signs also occurred in females of the same group, although the incidence was only 2 of 60. These neurological signs were quite identical to those observed in male ICR mice fed diets containing 15 and 30 ppm of MMC (Mitsumori et al., 198 1). However, there was a distinct difference in the time of onset of neurological signs between male B6C3Fl mice of the lo-ppm group and male ICR mice treated with 15 ppm or more; Signs were first observed in 1Oppm-fed B6C3Fl mice and 15- and 30-ppmfed ICR mice in Weeks 60,26, and 4, respectively. The difference in the time of onset between these treated groups may indicate that to reach a mercury concentration in the nervous system sufficient to produce neurological signs in animals treated with lower levels of MMC, longer treatment is needed. That no

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TABLE 2 INCIDENCE

OF EPITHELIAL

PROLIFERATIVE

LESIONS OFTHE

KIDNEY

IN

B6C3Fl MICE TREATED WITH MMC

Number of mice with lesions (%) Renal epithelial tumor Dose level (ppm)

Number of mice examined

Hyperplasia

Total

Male 0 0.4 2 10

60 60 60 60

0 0 0 14** (23)

0 0 1 (2) 16 (27)

Female 0 0.4 2 10

60 60 60 60

0 0 0 0

0 0 0

l(2)

Adenoma 0 0 1 (2) 5*(g) 0 0 0 (0) 1 (2)

Carcinoma 0 0 0 13**(22) 0 0 0 0

* p < 0.05: **p < 0.0 I (Fisher’s exact test), as compared to the controls.

neurological signs occurred in ICR mice fed 10 ppm MMC indicates that B6C3Fl mice are more sensitive than ICR mice to the chronic neurotoxic effects of MMC. Sensory neuropathy in the peripheral nervous system and neuronal necrosis in the cerebellum have been reported to occur frequently in rats given high doses of methylmercury for relatively short duration (Cavanagh and Chen, 197 1; Herman et al., 1973). The sensory neuropathy in rats was characterized by loss of nerve cells in the spinal ganglion and nerve fiber degeneration in the spinal dorsal root, spinal dorsal funiculus, and distal peripheral nerves such as the sciatic nerve (Herman et al., 1973). The cerebellar toxic lesion in rats was composed of necrosis and loss of small nerve cells in the granular cell layer (Cavanagh and Chen, 197 1). Cerebral lesions characterized by neuronal necrosis of the cortex have also been noted in rats (Miyakawa and Deshimaru, 1969), while the occurrence of the lesions was rare even in those animals that manifested neurological signs in response to administration of high doses of methylmercury. The morphological findings in these rat tissues were quite similar to those in B6C3Fl mice treated with 10 ppm

MMC in the present study. However, there were some differences in the anatomical distribution of neurotoxic changes between rats and mice given low doses of MMC. In the rats treated with 10 ppm MMC in our previous study (Mitsumori et al., 1984), toxic changes were detected only in the peripheral sensory nerve, not in the cerebrum and cerebellum. On the other hand, in B6C3Fl mice treated with 10 ppm MMC, toxic changes were observed in the peripheral sensory nerve, in the cerebrum, and in the cerebellum. These findings may indicate that B6C3Fl mice are more susceptible than rats to the induction of neurotoxic changes in the brain by the administration of low doses of MMC. Toxic nephropathy characterized by degeneration and necrosis ofthe proximal tubular epithelium has been observed in rats given relatively high doses of alkyl mercury compounds (Fowler, 1972). Such nephropathy has been shown to be aggravated by prolongation of the alkyl mercury treatment (Mitsumori et al., 1984). The chronic nephropathy observed in B6C3Fl mice in the present study was characterized by epithelial degeneration and regeneration of the proximal tubules and interstitial fibrosis, and was essen-

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FIG. 7. Renal carcinoma showing solid pattern composed of large epithelial cells with nuclear pleomorphism. Male B6C3Fl mouse in the IO-ppm group. H&E, X20.

tially the same as that observed in rats intoxicated with MMC (Mitsumori et al., 1984) and ICR mice treated with 10 ppm MMC or more (Mitsumori et al., 198 1; Hirano et al., 1986). This nephropathy significantly increased in incidence in male ICR mice treated with 2 ppm or more and in female ICR mice treated with 10 ppm, as compared to the control group (Table 3). In the present study, a similar increase in incidence of chronic nephropathy was observed in males of the 2-ppm group and in both males and females of the 10-ppm group. Therefore, it is evident that nephrotoxicity is an important parameter in the assessment of chronic toxicity of MMC in mice, and the susceptibility of B6C3Fl mice to renal toxicity is comparable to that of ICR mice. Tubular atrophy of the testis was observed in 47 of 60 males of the IO-ppm group in the present study. A similar increase in incidence of tubular atrophy was seen in ICR mice

treated with 10 ppm MMC (Hirano et al., 1986); however, the incidence (Table 3) was not high as compared to that in B6C3Fl mice of the IO-ppm group. Since a spermatogenic effect of methylmercury that results in reduced male fertility has been demonstrated in mice (Lee and Dixon, 1973), this tubular atrophy of the testis is considered to be a toxic effect of MMC. The incidence of ulcer in the glandular stomach (7/60) was also significantly increased in males of the lo-ppm group of the present study, as compared to the control group ( l/60). In contrast, such an ulcer was not observed in ICR mice treated with 10 ppm MMC in our previous study. There is no description of the occurrence of glandular stomach ulcer in other experimental animals intoxicated by alkyl mercury. However, the stomach ulcer observed in male B6C3Fl mice of the lo-ppm group was considered to be related to MMC treatment, since such an ulcer is a rare spontaneous le-

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FIG. 8. Focal tubular hyperplasia showing solid growth of large epithelial cells occluding the tubular lumen. Male B6C3FI mouse in the lo-ppm group. H&E, X40.

sion in aging B6C3F 1 mice (Ward et al., 1979). The morphological features of the renal epithelial tumors observed in male B6C3Fl mice of the IO-ppm group in the present study were similar to those induced in male ICR mice treated with 10 ppm MMC (Hirano et al., 1986). Renal epithelial tumors, including adenoma and carcinoma, in 16 of 60 lo-ppm-fed male B6C3Fl mice and 13 of 59 lo-ppm-fed male ICR mice. Since the renal tumor was first microscopically observed at Week 60 in the B6C3Fl mice and at Week 58 in the ICR mice, the effective numbers of males in the IO-ppm groups of B6C3Fl and ICR mice were calculated to be 57 and 26, respectively. However, 28 of the 57 male B6C3Fl mice became moribund or died during Weeks 60 to 100, and their life span was clearly shortened because of the severe neurotoxic effects. Therefore, the true effective number of male B6C3F 1 mice in the 10-ppm

group is considered to be less than 30 and is approximately comparable to that of male ICR mice in the lo-ppm group. On the basis of these findings, it is concluded that no difference exists in the renal carcinogenic potential of MMC between B6C3Fl and ICR male mice. The guidelines for chemical oncogenicity studies provided by the National Toxicology Program (1984) state that an appropriate maximum tolerated dose (MTD) for an oncogenicity study should be the maximum dose necessary to elicit signs of minimal toxicity without significantly altering the animal’s normal life span through effects other than carcinogenicity. In the present study, the highest dose level ( 10 ppm) clearly affected survival of B6C3Fl males in which the neurotoxic and oncogenic effects were seen, though B6C3Fl mice received the same dosage as ICR mice in our previous study. Therefore, the present study was considered

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MICE

TABLE 3 SUMMARY OFTHE PREVIOUS ~-YEAR FEEDING STUDY OF MMC IN ICR MICE Do= group (ppm) Male Findings

0

0.4

Number of mice examined Incidence of neurologic signs Mortality Body weight Food consumption MMC intake (mg/kg/day) Incidence of histological findings Chronic nephropathy Testicular tubular atrophy Renal tubular hyperplasia Renal adenoma Renal carcinoma

60 0

60 0 NS” NS NS 0.038

4 1 0 1 0

Female 2

10

60 0 NS NS NS 0.188

60 0 NS NS NS 0.906

17 5 1 0 0

47** II** 13** 3 10**

3 3 0 0 0

0

0.4

60 0

60 0 NS NS NS 0.032

60 0 NS NS NS 0.144

60 0 NS NS NS 0.785

13 0 0 0

12 0 0 0

26* 0 0 0

-

9 0 0 0

2

10

’ NS, no significant change; -, not available. * p < 0.05: **p < 0.01 (Fisher’s exact test), as compared to the control.

to have served as an adequate chronic toxicity study but not as a definitive oncogenicity study for MMC, since the criteria for the proper selection of an MTD were not satisfied. In other words, it was indicated that the adequate dose between 10 and 2 ppm should have been used as the highest dose level for the purposes of evaluating the carcinogenicity of MMC in B6C3Fl mice. In conclusion, the present study indicates that B6C3Fl mice are more sensitive to the neurotoxic effects of MMC than are ICR mice, while the susceptibility of B6C3Fl mice to renal carcinogenicity is comparable to that of ICR mice. In addition, B6C3Fl mice are considered to be more appropriate than ICR mice for carcinogenicity studies in mice, since the survival rate at Week 104 of control ICR mice was remarkably lower than that of control B6C3Fl mice in the present study. Further studies are necessary to clarify which strain/hybrid is the most suitable for safety evaluations using mice. REFERENCES CAVANAGH, J. B., AND CHEN, F. C. K. (1971). The effects of methyl-mercury-dicyandiamide on the pe-

ripheral nerves and spinal cord of rats. Acta Neuropa19,208-2 15. FOWLER, B. A. (1972). The morphologic effects of dieldrin and methyl mercuric chloride on pars recta segments of rat kidney proximal tubules. Amer. J. Pathol. 69,163-178. HERMAN, S. P., KLEIN, R., TALLEY, F. A., AND KRIGMAN, M. R. (1973). An ultrastructural study of methylmercury-induced primary sensory neuropathy in the rat. Lab. Invest. 28, 104-l 18. HIRANO, M., MITSUMORI, K., MAITA, K., AND SHIRASU, Y. (1986). Further carcinogenicity study on methylmercury chloride in ICR mice. Japan. J. Vet. Sci. 48, 127-135. HIRANO, M., UEDA, H., MITSUMORI, K., MAITA, K., AND SHIRASU, Y. (1988). Hormonal influence on carcinogenicity of methylmercury in mice. Japan. J. Vet. thol.

Sci. 50,886-893.

JAEGER, B. (1984). Pesticide

Assessment Guidelines. Subdivision F. Hazard Evaluation: Human and Domestic Animals. U.S. Environmental Protection

Agency, Washington, DC. LEE, I. P., AND DIXON, R. L. (1973). Effects of mercury on mouse spermatogenesis studied by cell separation and serial mating (abstract). Toxicol. Appl. Pharmacol. 25,464. MITSUMORI, K., MAITA, K., SAITO, T., TSUDA, S., AND SHIRASU, Y. (1981). Carcinogenicity of methylmercury chloride in ICR mice: Preliminary note on renal carcinogenesis. Cancer Lett. 12,305-3 10. MITSUMORI, K., MAITA, K., AND SHIRWJ, Y. (1984). Chronic toxicity of methylmercury chloride in rats: Pathological study. Japan. J. Vet. Sci. 46,549-557.

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MITSUMOR~, K.. TAKAHASHI, K., MATANO, 0.. GOTO, S., AND SHIRASU, Y. (1983). Chronic toxicity of methylmercury chloride in rats: Clinical study and chemical analysis. Japan. .I. Vet. Sci. 45,141-151. MIYAKAWA, T., AND DESHIMARU, M. (1969). Electron microscopical study of experimentally induced poisoning due to organic mercury compound-Mechanism of development of the morbid change. Acta Neuroparhol. 14, 126-136. National Toxicology Program, Board of Scientific Counselors (1984). Report of the NTP Ad Hoc Panel on Chemical Carcinogenesis Testing and Evaluation.

ET AL.

U.S. Department of Health and Human Services, Puhlit Health Service. RAO, G. N., BIRNBAUM, L. S., COLLINS, .I. J., TENNANT, R. W., AND SKOW, L. C. (1988). Mouse strains for chemical carcinogenicity studies: Overview of a workshop. Fzcndam. Appl. Toxicol. 10, 385-394.

WARD, J. M., GOODMAN, D. G., SQUIRE, R. A., CHU, K. C., AND LINHART, M. S. (1979). Neoplastic and nonneoplastic lesions in aging (C57BL/6N X C3H/ HeN) Fl (B6C3Fl) mice. J. Natl. Cancer Inst. 63, 849-854.

Chronic toxicity and carcinogenicity of methylmercury chloride in B6C3F1 mice.

A 2-year feeding study of methylmercury chloride (MMC: 0, 0.4, 2, or 10 ppm) was conducted in B6C3F1 mice (60 mice of each sex/group) to compare chron...
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