Vitelline cyst in the rat ileum.

Congenital vitelline duct anomalies other than Meckel's diverticulum are rare in animals. A cyst of approximately 8 mm in diameter was observed on the antimesenteric surface of the ileal serosa in a 10-week-old female Crl:CD(SD) rat. Microscopically, the cyst closely resembled the ileum, but it did not communicate with the ileal lumen. We diagnosed this case as a vitelline cyst derived from the vitelline duct based on the location where it developed and its histological behavior. In rats, only Meckel's diverticulum has been reported with a congenital anomaly of the vitelline duct, and no other spontaneous anomalies including a vitelline cyst have been reported. This case may be the first report concerning a vitelline cyst in the rat ileum.

EpCAM, a potential therapeutic target for esophageal squamous cell carcinoma.

BACKGROUND: Molecular-targeted drugs are not available for esophageal squamous cell carcinoma (ESCC), which has a poor prognosis. We investigated the clinicopathological significance of epithelial cell adhesion molecule (EpCAM) expression and the utility of EpCAM as a potential therapeutic target. METHODS: The relationship between EpCAM expression and clinicopathological factors was examined by immunohistochemistry in 74 patients with resectable ESCC. A total of ten ESCC cell lines were analyzed for EpCAM expression. The effects of EpCAM knockdown in TE4, TE10, and TE14 cells were examined with regard to cell proliferation and gene expression in vitro and tumor growth in vivo. The antitumor effect of catumaxomab in ESCC cell lines was examined. RESULTS: EpCAM overexpression was associated with poor survival in ESCC patients (P = 0.026). Multivariate Cox regression analysis showed that EpCAM overexpression was a significant and independent prognostic factor for surgically treated ESCC (P = 0.004). TE4 and TE10 cells showed high EpCAM expression, in contrast to TE14. EpCAM siRNA knockdown in TE4 and TE10 cells downregulated CCND1 and CCNE2 and suppressed cell proliferation. Low EpCAM expression reduced tumorigenesis; TE4 cells initiated tumorigenesis in seven of the ten mice injected, whereas shRNA knockdown resulted in smaller tumors in two of ten mice at 6 weeks after transplantation. Concentration- and time-dependent antitumor effects of catumaxomab were observed in TE4 and TE10 cells. CONCLUSIONS: EpCAM overexpression is an independent prognostic factor for surgically treated ESCC. EpCAM contributes to cell proliferation and tumorigenesis and may be a useful therapeutic target for ESCC.

Neutrophil elastase inhibitor improves survival rate after ischemia reperfusion injury caused by supravisceral aortic clamping in rats.

BACKGROUND: Sivelestat sodium hydrate is a specific neutrophil elastase inhibitor effective in acute lung injury (ALI) associated with systemic inflammatory response syndrome. Bowel ischemia reperfusion injury (IRI) induced by supravisceral aortic clamping is associated with an excessive systemic inflammatory response, resulting in remote organ damage, including ALI. In this study, we investigated whether sivelestat can attenuate neutrophil sequestration in the lung, alleviate ALI, and improve survival in a rat bowel IRI model. METHODS: Adult male Sprague-Dawley rats underwent bowel IRI induced by supravisceral aortic clamping and were randomly assigned to receive sivelestat or saline (control) and monitored for survival. We randomly assigned other rats to undergo laparotomy alone (sham operation), IRI alone, or IRI and sivelestat treatment. We evaluated blood samples for organ function, cytokine levels, and neutrophil elastase activity after reperfusion. Organs were analyzed histologically. We also determined lung injury in another set of rats. RESULTS: Bowel IRI induced a significant increase in serum variables indicative of organ function, cytokine concentrations, neutrophil elastase activity, and lung permeability and edema, which reflected the presence of both systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome. Treatment with sivelestat significantly improved survival rate, lung permeability and edema, and significantly decreased levels of creatinine, interleukin 6, interleukin 10, and neutrophil elastase activity. Histological studies showed that sivelestat-treated rats had less bowel IRI-induced damage to lung and liver tissue than controls. CONCLUSION: In a rat model, administration of sivelestat attenuated the effects of bowel IRI induced by supravisceral aortic clamping, and improved the survival rate.

Development of a repeated-dose liver micronucleus assay using adult rats (II): further investigation of 1,2-dimethylhydrazine and 2,6-diaminotoluene.

Detecting genotoxicity in the liver is considered an effective approach for predicting hepatocarcinogenicity, as many genotoxic chemicals in vivo may act as hepatocarcinogens in rodents. Here, a genotoxic rodent hepatocarcinogen, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), and a genotoxic (Ames positive) noncarcinogen, 2,6-diaminotolunene (2,6-DAT), were administered orally to rats for up to 28 days, and liver samples were then examined in a repeated-dose liver micronucleus (MN) assay, and additionally tested in the bone marrow (BM) MN assay concurrently. We recently established a simple method to isolate hepatocytes without in situ liver perfusion procedures, and applied this method in the liver MN assay. As a result, 1,2-DMH increased the proportion of micronucleated hepatocytes in both a dose- and duration-dependent manner at relatively low-dose levels that are routinely used in repeated-dose toxicity studies. In contrast to 1,2-DMH, 2,6-DAT did not have a detectable effect. In addition to these two chemicals, two genotoxic rodent hepatocarcinogens, diethylnitrosamine and 2,4-diaminotoluene, which gave positive responses in the liver MN assay in our previous investigation [Narumi et al., Mutat. Res. 747 (2012) 234-239], were subjected to the BM MN assay and histopathological evaluation. All four test chemicals gave negative responses in the BM MN assay. Furthermore, the three hepatocarcinogens displayed hepatotoxicity, including hepatocellular hypertrophy and anisokaryosis, but no abnormal findings were observed in the liver of rats treated with 2,6-DAT. Taken together, the present results indicate that the liver MN assay is effective for predicting hepatocarcinogenicity and may be integrated into repeated-dose toxicity studies without disturbing routine examinations, such as histopathology. Furthermore, with repeat-dose treatment protocols, our findings indicate that the liver MN assay is superior to the BM MN assay for detecting genotoxic or carcinogenic chemicals in rats.

Detection of hepatocarcinogens by combination of liver micronucleus assay and histopathological examination in 2-week or 4-week repeated dose studies.

BACKGROUND: Currently, revisions to the ICH S1 guidance on rodent carcinogenicity testing are being proposed. Application of this approach would reduce the use of animals in accordance with the 3Rs principles (reduce/refine/replace). The method would also shift resources to focus on more scientific mechanism-based carcinogenicity assessments and promote safe and ethical development of new small molecule pharmaceuticals. In the revised draft, findings such as cellular hypertrophy, diffuse and/or focal cellular hyperplasia, persistent tissue injury and/or chronic inflammation, preneoplastic changes, and tumors are listed as histopathology findings of particular interest for identifying carcinogenic potential. In order to predict hepatocarcinogenicity of test chemicals based on the results from 2- or 4-week repeated dose studies, we retrospectively reanalyzed the results of a previous collaborative study on the liver micronucleus assay. We focused on liver micronucleus induction in combination with histopathological changes including hypertrophy, proliferation of oval cells or bile duct epithelial cells, tissue injuries, regenerative changes, and inflammatory changes as the early responses of hepatocarcinogenesis. For these early responses, A total of 20 carcinogens, including 14 genotoxic hepatocarcinogens (Group A) and 6 non-liver-targeted genotoxic carcinogens (Group B) were evaluated. RESULTS: In the Group A chemicals, 5 chemicals (NPYR, MDA, NDPA, 2,6-DNT, and NMOR) showed all of the 6 early responses in hepatocarcinogenesis. Five chemicals (DMN, 2,4-DNT, QUN, 2-AAF, and TAA) showed 4 responses, and 4 chemicals (DAB, 2-NP, MCT, and Sudan I) showed 3 responses. All chemicals exhibited at least 3 early responses. Contrarily, in the Group B chemicals (6 chemicals), 3 of the 6 early responses were observed in 1 chemical (MNNG). No more than two responses were observed in 3 chemicals (MMC, MMS, and KA), and no responses were observed in 2 chemicals (CP and KBrO3). CONCLUSION: Evaluation of liver micronucleus induction in combination with histopathological examination is useful for detecting hepatocarcinogens. This assay takes much less time than routine long-term carcinogenicity studies.

Vascular smooth muscle lipofuscinosis occurring predominantly in veins of a cynomolgus monkey (Macaca fascicularis).

A 6-year-old male cynomolgus monkey showed chronic wasting. No gross abnormalities were observed in necropsy except for changes secondary to wasting. Microscopic examination revealed pigment granules deposition in systemic smooth muscles. They were observed as brown or basophilic in hematoxylin and eosin stain, and were positive for periodic acid-Schiff, Schmorl and Ziehl-Neelsen. Ultrastructurally, they consisted of residual bodies surrounded with varying amounts of solitary ribosomes. Thus, these granules were considered as lipofuscin. Unlike brown bowel syndrome in humans, the pigment granules were distributed systemically not only in the digestive tract but also in the blood vessels predominantly in the veins. To our knowledge, this is the first report on vascular smooth muscle lipofuscinosis occurring predominantly in the veins of primates.

Needle-shaped amyloid deposition in rat mammary gland: evidence of a novel amyloid fibril protein.

Amyloidosis is an extremely rare event in rats. In this study, we report that lipopolysaccharide binding protein (LBP) is the most likely amyloidogenic protein in rat mammary amyloidosis. Histologically, corpora amylacea (CA) and stromal amyloid (SA) were observed in rat mammary glands, and needle-shaped amyloid (NA) was also observed on the surface or gap of CA and SA. Following surveillance in aged rats, NA was observed in 62% of mammary tumours, 25% of male mammary glands and 83% of female mammary glands. Proteomic analysis showed that lactadherin was a major constitutive protein of CA and SA, and both were positive following immunohistochemistry with anti-lactadherin antibodies. In the same analysis, LBP was detected as a prime candidate protein in NA, and NA was positive following immunohistochemistry and immunoelectron microscopy with anti-LBP antibody. Furthermore, synthetic peptides derived from rat LBP formed amyloid fibrils in vitro. Overall, these results provide evidence that LBP is an amyloid precursor protein of NA in rat mammary glands.

Histologic evaluation of the diversity of epidermal laminae in hooves of horses without clinical signs of laminitis.

OBJECTIVE: To evaluate the histologic diversity of epidermal laminae in hooves from horses without clinical signs of laminitis. SAMPLE POPULATION: Formalin-fixed samples of stratum internum obtained from the mid region of the dorsal aspect of the hoof wall from the forelimbs of 35Thoroughbred cadavers (including foals [n = 9], yearlings [5], 2 year olds [6], racing horses [5], and mares [10]). PROCEDURES: Paraffin-embedded laminar tissues were stained with H&E for the evaluation of architectural variety of primary epidermal laminae (PEL) and secondary epidermal laminae (SEL). For detection of cytokeratin (CK) expression in epidermal laminae, immunohisto-chemical staining was performed by use of anti-CK14 and anti-CK8.12 antibodies. RESULTS: The morphology of the PEL, SEL, and tips of PEL was classified into 3, 5, and 3 patterns, respectively. Differences in the predominant type of SEL depended on their location with respect to the laminar interface. In SEL attached to the sides of PEL, the basal cells were immunoreactive to CK14 and CK8.12, which was interpreted as a normal pattern. In some SEL at the tips of PEL, the suprabasal cells expressed CK14, CK8.12, or both, which constituted a hyperplastic pattern. CONCLUSIONS AND CLINICAL RELEVANCE: The histologic diversity of epidermal laminae from hooves of Thoroughbreds was attributable to the combined morphology of PEL and SEL. Detection of hyperplastic changes in the laminar interface does not justify a diagnosis of laminitis because such changes can develop independent of clinical disease. The classification system used here should aid investigators in making a more accurate histologic evaluation of laminae.

Expression of monocarboxylate transporter 1 (MCT1) in the dog intestine.

In this study, the expression and distribution of monocarboxyolate transporter 1 (MCT1) along the intestines (duodenum, jejunum, ileum, cecum, colon and rectum) of dogs were investigated at both the mRNA and protein levels. The expression of MCT1 protein and its distribution were confirmed by Western blotting and immunohistochemical staining using the antibody for MCT1. We identified mRNA coding for MCT1 and a 43-kDa band of MCT1 protein in all regions from the duodenum to the rectum. Immunoreactive staining for MCT1 was also observed in epithelial cells throughout the intestines. MCT1 immunoreactivity was greater in the large intestine than in the small intestine. MCT1 protein was predominantly expressed on the basolateral membranes along intestinal epithelial cells, suggesting that MCT1 may play an important role in lactate efflux and transport of short-chain fatty acids (SCFAs) to the bloodstream across the basolateral membranes of the dog intestine.

Immunohistochemical evaluation of canine ovarian tumors.

Canine ovarian tumors (epithelial tumor, sex-cord stromal tumor, germ cell tumor) classifying into 9 histological types were examined immunohistochemically using placental alkaline phosphatase (PLAP), cytokeratin7 (CK7), desmin, S100, AE1/AE3, inhibin alpha, vimentin, and alfa feto-protein (AFP). The papillary and tubular types observed in epithelial tumors were immunoreactive for desmin and AE1/AE3. The papillary type was also immunoreactive for PLAP and CK7. The solid type, nest type, cord type, palisade type, cystic type and spindle type, which were observed in sex-cord stromal tumors, showed a positive immunoreaction for S100 but little or no positive immunoreaction for inhibin alpha with an exception of positive result in the palisade type. Most of the sex-cord stromal tumors were AE1/AE3-positive except for the palisade type. In the cobblestone type observed in germ cell tumors, only vimentin and AFP were positive. The present study elucidated the detailed histological and immunohistochemical characteristics of canine ovarian tumors.

Immunohistochemical evaluation of canine ovarian cysts.

To clarify the immunohistochemical characteristics of canine ovarian cysts, 109 canine ovarian cysts (57 cysts of subsurface epithelial structures: SES, 26 graafian follicle cysts, 12 cystic rete ovarii and 14 cysts difficult to classify morphologically) were examined regarding their lining cells immunohistochemically using antibodies against placental alkaline phosphatase (PLAP), S100, inhibin alpha, desmin and AE1/AE3. Both cysts of SES and cystic rete ovarii had a positive immunoreaction to desmin and AE1/AE3, whereas all cysts all but graafian follicle cysts were negative for inhibin alpha. PLAP-positive immunoreaction was observed only in cysts of SES. Graafian follicle cysts had a positive immunoreaction to inhibin alpha, but were negative for PLAP, desmin and AE1/AE3. Fourteen cysts were difficult to classify morphologically because these cysts had single-squamous lining cells and lacked other morphological characteristics. However, these unclassified cysts were immunohistochemically divided into two groups, including positive and negative cysts, by the reactivity of PLAP. The PLAP-positive cysts were considered large cysts of SES. These results suggest that PLAP was a useful marker for classification of cysts of SES, although cysts originating from SES are not always positive for this antigen.

Jejunal fibroplasia in a rat.

A jejunal nodular mass was identified in an aging rat. Histologically, the boundaries between the lesion and surrounding normal tissue as well as between the inner circular muscle and outer longitudinal muscle were indistinct. The lesion consisted of abundant eosinophilic matrix and cells with a large round to oval nucleus and indistinct cytoplasm. There was no characteristic proliferating pattern, nuclear polymorphism and a low mitotic figure count. Masson's trichrome stain revealed that the intestinal smooth muscles were replaced by the abundant collagen fiber. Immunohistochemistry revealed that the cells with a large round to oval nucleus were labeled with anti-vimentin antibody and not with anti-α smooth muscle actin antibody, suggesting that these cells were fibroblasts. The mass was diagnosed as jejunal fibroplasia.

Lymphocytic adrenal medullitis and lymphocytic thyroiditis in a laboratory beagle dog.

Lymphocytic adrenal medullitis characterized by inflammation and atrophy in the medulla of the bilateral adrenal glands was observed in an 18-month-old male laboratory beagle dog. It might be that the present lymphocytic adrenal medullitis is an autoimmune-mediated disease as the histological characteristics are consistent with an autoimmune pathogenesis. However, the actual cause remains unclear as the existence of serum autoantibodies against the adrenal medulla could not be confirmed. Although this dog also contracted lymphocytic thyroiditis along with serum thyroglobulin autoantibodies, indicating that the thyroiditis occurred with an autoimmune basis; the relation between the adrenal medullitis and thyroiditis is unknown.

Evaluation of the repeated-dose liver micronucleus assay with p-dimethylaminoazobenzene.

The micronucleus induction by p-dimethylaminoazobenzene (DAB), a genotoxic rat liver carcinogen, was assessed in the liver and bone marrow of young adult rats after the repeated administration of DAB for 14 (Lab. 1) and 28 (Lab. 2) days. Three dose levels, 25, 50 and 100mg/kg/day, were used for the investigations in both labs. The frequency of micronucleated hepatocytes was significantly increased in a dose-dependent manner after the repeated administration of DAB at 50mg/kg/day or more for 14 and 28 days. Similarly, the frequency of micronucleated immature erythrocytes in the bone marrow was increased after the repeated administration of DAB at 100mg/kg/day for 14 and 28 days. These results indicate that the repeated-dose liver micronucleus assay allowed for the detection of micronucleus induction by DAB, and that the lowest detectable dose for micronucleus induction in the liver was lower than in the bone marrow. Thus, the repeated-dose liver micronucleus assay using young adult rats is considered suitable for the detection of micronucleus induction by liver carcinogens, such as DAB.

Evaluation of in vivo genotoxicity by thioacetamide in a 28-day repeated-dose liver micronucleus assay using male young adult rats.

The repeated-dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens and can be integrated into general toxicological studies. In this study, thioacetamide (TAA) was tested in 14- and 28-day RDLMN assays to assess the performance of the assay. The test substance, TAA, was administered orally to 6-week-old male Crl:CD (SD) rats once daily for 14 or 28 days at a dosage of 5, 10 or 20mg/kg/day. Hepatocytes were collected approximately 24h after the last TAA administration, and the incidence of micronuclei was assessed. In this study, bone marrow micronucleus assays were also conducted in the same animals. The 14- and 28-day RDLMN assays indicated that none of the TAA dosages significantly increased the proportion of micronucleated hepatocytes. Bone marrow micronucleus assays with TAA also provided negative results. It is known that TAA is a liver carcinogen in mice and rats. In the previous genotoxic studies, the Ames test and the chromosomal aberration test using CHL/IU cells have yielded negative results [1-4]. The liver micronucleus assay using young adult rats singly dosed with TAA (75 and 150mg/kg) also produced negative results [5]. TAA gave positive results only in the mouse bone marrow micronucleus assays [6,7].

Repeated-dose liver micronucleus test of 4,4'-methylenedianiline using young adult rats.

Liver micronucleus (MN) tests using partial hepatectomized rats or juvenile rats have been shown to be useful for the detection of hepatic carcinogens. Moreover, Narumi et al. established the repeated-dose liver MN test using young adult rats for integration into general toxicity. In the present study, in order to examine the usefulness of the repeated-dose liver MN test, we investigated MN induction with a 14 or 28 day treatment protocol using young adult rats treated with 4,4'-methylenedianiline (MDA), a known hepatic carcinogen. MDA dose-dependently induced micronuclei in hepatocytes in 14- and 28-day repeated-dose tests. However, although statistically significant increases in micronuclei were observed in bone marrow cells at two dose levels in the 14-day study, there was no dose response and no increases in micronuclei in the 28-day study. These results indicate that the evaluation of genotoxic effects using hepatocytes is effective in cases where chromosomal aberrations are not clearly detectable in bone marrow cells. Moreover, the repeated-dose liver MN test allows evaluation at a dose below the maximum tolerable dose, which is required for the conventional MN test because micronucleated hepatocytes accumulate. The repeated-dose liver MN test employed in the present study can be integrated into the spectrum of general toxicity tests without further procedural modifications.

Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone.

Repeated-dose liver and gastrointestinal tract micronucleus assays with CI Solvent Yellow 14 (Sudan I) using young adult rats.

The in vivo genotoxicity of CI Solvent Yellow 14 (Sudan I) was examined using repeated-dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats. Sudan I is a mono-azo dye based on aniline and 1-amino-2-hydroxynaphthalene. This dye was demonstrated as a rat liver carcinogen in a National Toxicology Program (NTP) bioassay, and genotoxicity was noted in a rat bone marrow micronucleus (BMMN) assay. In the present study, Sudan I was administered orally to rats for 14-days, and the MN frequency in the liver, stomach, colon, and bone marrow were analyzed. The frequency of micronucleated hepatocytes (MNHEPs) was not significantly increased by the administration of the Sudan I. Gastrointestinal tract MNs were also not induced. However, in the BMMN assay, a significant increase in micronucleated immature erythrocytes (MNIMEs) was observed in a dose-dependent manner. While Sudan I has been reported to lack hepatic genotoxicity, it has also exhibited tumor-promoting activities. These results are consistent with the lack of induction of MN in the hepatocytes. The lack of MN induction in cells of the gastrointestinal tract was also logical because azo-compounds are reported to be unlikely to induce DNA damage in the rat gut. The repeated-dose rat liver and gastrointestinal tract MN assays have the potential to be used in the evaluation of the genotoxicity of a chemical in each organ in accordance with its mode of action.

Repeated dose liver and gastrointestinal tract micronucleus assays using N-methyl-N'-nitro-N-nitrosoguanidine in young adult rats.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a direct-acting mutagen that induces tumors in the glandular stomach, but not in the liver or colon, of rats after oral administration. To evaluate the performance of repeated dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats, MNNG was administered by oral gavage to male CD (SD) rats aged 6 weeks at doses of 0 (vehicle; 2.5% DMSO aqueous solution), 3.125, 6.25, 12.5, and 25mg/kg/day once daily for 14 and 28 days, and the MN frequencies were examined in the hepatocytes, glandular stomach cells, and colonic cells. The MN induction in immature erythrocytes in the bone marrow of these animals was also simultaneously evaluated. The frequencies of micronucleated (MNed) glandular stomach cells were significantly increased in all MNNG treatment groups in a dose-dependent manner in both repeated dose studies. In contrast, the frequencies of MNed hepatocytes and colonic cells were not significantly increased compared to the vehicle control. In the bone marrow, a small but significant increase in the frequency of MNed immature erythrocytes was observed only at the highest dose in the 28-day study. Since a clear positive result in the glandular stomach agrees with the tissue specificity of tumor induction by this chemical, the MN assay with the glandular stomach, which is a direct contact site with high concentrations of test substances administered by oral gavage, may be useful for detecting genotoxic compounds that are short-lived in vivo, such as MNNG.

Assessment of methyl methanesulfonate using the repeated-dose liver micronucleus assay in young adult rats.

A repeated-dose liver micronucleus assay using young adult rats was conducted with methyl methanesulfonate (MMS) as a part of a collaborative study supported by the Collaborative Study Group for the Micronucleus Test/the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. MMS is a classical DNA-reactive carcinogen, but it is not a liver carcinogen. In the first experiment (14-day study), MMS was administered per os to 6-week-old male Crl:CD (SD) rats every day for 14 days at a dose of 12.5, 25, or 50mg/kg/day. In the second experiment (28-day study), 6-week-old male SD rats were treated with MMS at 7.5, 15, or 30mg/kg/day for 28 days, because the highest dose used in the 14-day study (50mg/kg/day) caused mortality. Hepatocyte and bone marrow cell specimens were prepared on the day after the final dose. The frequency of micronucleated hepatocytes (MNHEPs) in the liver and that of micronucleated immature erythrocytes (MNIMEs) in the bone marrow were evaluated. Exposure to 50mg/kg/day MMS for 14 days resulted in an increased frequency of MNHEPs, but MMS had no effect on the frequency of MNHEPs in the rats exposed to the chemical for 28 days at doses up to 30mg/kg/day. MMS induced MNIMEs production at doses of 25 and 50mg/kg/day in the 14-day study and at doses of 15 and 30mg/kg/day in the 28-day study. Overall, the effect of MMS on the frequency of MNHEPs was considered to be equivocal.