LEUCOVORIN AND MAXIMUM TOLERATED DOSE TOXICITY OF METHOTREXATE IN RATS

High-dose methotrexate (HD-MTX) is widely used in combination chemotherapy and can be handled without life-threatening toxicity in combination with leucovorin (LV) rescue. However, in an experimental animal modelfortesting of short-term HD-MTX effects in anesthetized rats, the authors previously demonstrated intolerable toxicity and death within a few hours in some animals. Serum levels were below levels routinely found in patients on HD-MTX treatment. This study was aimed at disclosure of possible mechanisms for acute toxicity of MTX in rats. The previously determined maximum tolerated dose of 5 g/kg MTX was used as the test dose. The animals that died showed sudden reduction in heart rate and blood pressure. LV, 1 g/kg infused immediately prior to MTX, changed MTX elimination kinetics, but did not change the acute toxicity. The data of this study together with additional evidence obtained in the experimental model, suggest that MTX acute toxicity may not be related to its antiproliferative effect, but rather to perturbation of endothelial cell and platelet function.     

Variability in dose intensity of high-dose methotrexate for nonmetastatic osteosarcoma

The authors evaluated their ability to maintain planned dosing schedules for high-dose methotrexate (HD-MTX) in patients with nonmetastatic osteosarcoma. Twenty-seven patients who received therapy according to 2 POG protocols (8651 and 9351), both of which included HD-MTX (12 g/m(2)/week for 2 consecutive weeks), between 1988 and 1998 were studied. Significantly fewer HD-MTX infusions were given on the second week to patients treated on POG 9351 (33 vs. 93%; p < .0001). The hydration guidelines were identical and there was no difference in peak serum MTX levels either within or between protocols. Differences in the administration of combination chemotherapy in 9351 compared to 8651 may have contributed to the increased toxicity associated with HD-MTX on 9351, although this is speculative. The use of HD-MTX should be carefully planned so that it does not decrease its dose intensity or that of other effective agents.     

大剂量甲氨蝶呤治疗急性淋巴细胞白血病

目的研究3g/m2和5g/m2甲氨蝶呤(MTX)治疗急性淋巴细胞白血病(ALL)的血、脑脊液MTX浓度和不良反应。方法ALL患儿43例共接受98例次MTX3g/(m2·次)或5g/(m2·次)治疗,对两剂量组进行MTX血药质量浓度、脑脊液浓度及不良反应比较。结果1.MTX44、66h血药质量浓度与23hMTX血药质量浓度明显相关(P<0.05);2.不同个体间及同一个体不同时间使用同一给药方案血药质量浓度、脑脊液浓度水平差异较大;3.两剂量组不良反应发生率无明显差异(P>0.05),骨髓抑制、肝功能损害的MTX血药质量浓度无明显差异(P>0.05)。结论对于标危、高危ALL分别采用3、5g/(m2·次)的剂量是合理的,无严重不良反应发生。     

Influence of high-dose methotrexate therapy (HD-MTX) on glomerular and tubular kidney function

BACKGROUND: The present investigation was intended to further clarify the mechanisms involved in renal dysfunction following high-dose methotrexate (HD-MTX) treatment. PATIENTS AND METHODS: Fifty eight predominately pediatric patients [39 male, 19 female; mean age 12.3 years (range 2.2-34.1)] suffering from acute lymphoblastic leukemia (ALL, n = 28), Non Hodgkins lymphoma (NHL, n = 13), osteosarcoma (n = 8), malignant brain tumor (n = 6), or an ALL relapse (n = 3), were prospectively examined. In the course of 220 infusions of HD-MTX, glomerular and tubular renal function was determined by measuring proteinuria and glomerular filtration rate (GFR), as well as renal excretion of alpha-1-microglobulin (AMG) and N-acetyl-beta-D-glucosaminidase (NAG). It was investigated whether there were differences in MTX toxicity in dependence on the administered dose (1, 5, or 12 g/m(2) BSA), on the combination with other cytostatic agents (ifosfamide or cyclophosphamide), on the metabolism of MTX into 7-OH-MTX, and on pre-treatment with MTX. RESULTS: The administration of HD-MTX has no direct tubulotoxic effect. The disturbance in glomerular function was dose dependently and indicated by an increase in proteinuria as well as by a decrease in GFR; all changes were completely reversible and did not correlate to the metabolism of MTX to 7-OH-MTX. Increasing the number of MTX therapeutic cycles did not increase the nephrotoxicity of MTX. CONCLUSION: MTX is not directly tubulotoxic. Its side effects on glomeruli are usually without clinical relevance.     

VARIABILITY IN DOSE INTENSITY OF HIGH-DOSE METHOTREXATE FOR NONMETASTATIC OSTEOSARCOMA

The authors evaluated their ability to maintain planned dosing schedules for high-dose methotrexate (HD-MTX) in patients with nonmetastatic osteosarcoma. Twenty-seven patients who received therapy according to 2 POG protocols (8651 and 9351), both of which included HD-MTX (12 g/m 2 /week for 2 consecutive weeks), between 1988 and 1998 were studied. Significantly fewer HD-MTX infusions were given on the second week to patients treated on POG 9351 (33 vs. 93%; p < .0001). The hydration guidelines were identical and there was no difference in peak serum MTX levels either within or between protocols. Differences in the administration of combination chemotherapy in 9351 compared to 8651 may have contributed to the increased toxicity associated with HD-MTX on 9351, although this is speculative. The use of HD-MTX should be carefully planned so that it does not decrease its dose intensity or that of other effective agents.     

儿童急性白血病应用大剂量甲氨蝶呤严重并发症3例临床分析及文献复习

目的探讨儿童白血病（ALL）应用大剂量甲氨蝶呤（HD-MTX）严重并发症的临床特点、发生机制及预防处理措施。方法总结3例在本院确诊并按ALL治疗方案规范化疗的儿童急性淋巴细胞白血病应用HD．MTX后出现严重并发症病例的临床特点并复习文献。结果3例患儿均为学龄期儿童、急性淋巴细胞白血病应用HD．MTx（5g／m2）期间发生严重高甲氨蝶呤血症,治疗期间严密监测血药浓度,持续水化、碱化以及多种方式四氢叶酸钙解救但甲氨蝶呤排泄延迟,2～3周后血清甲氨蝶呤浓度仍明显高于中毒浓度,化疗后2周均合并严重骨髓抑制,2例出现表皮松解、1例消化道溃疡,均因继发感染及多脏器损害并发症死亡。结论甲氨蝶呤体内的代谢受到多种因素影响,持续高血药浓度可造成严重并发症,对儿童ALL应用HD．MTX应根据细胞免疫学表型、细胞遗传学及甲氨蝶呤静脉滴注后不同时段的血药浓度及药物代谢酶相关基因情况采用个体化治疗。     

Methotrexate induces germ cell apoptosis and impairs spermatogenesis in a rat

Purpose

The primary toxic effects of methotrexate (MTX) are myelosuppression and/or intestinal mucositis. The objective of the present study is to investigate the effect of MTX on germ cell apoptosis and spermatogenesis in a rat.

Methods

Male Sprague–Dawley rats were divided into three experimental groups: control rats treated with vehicle; MTX-2 rats treated with one dose (20 μg/kg) of MTX given IP and killed on the second day; and MTX rats treated with IP MTX (20 μg/kg) and killed on day 4. Johnsen’s criteria and the number of germinal cell layers in the testes were used to categorize the spermatogenesis. TUNEL assay was used to determine germ cell apoptosis. Western blotting was used to determine Bax and Bcl-2 protein levels. Statistical analysis was performed using the non-parametric Kruskal–Wallis ANOVA test, with p less than 0.05 considered statistically significant.

Results

On day 2, MTX-treated animals demonstrated minimal changes in the histological parameters of spermatogenesis, but germ cell apoptosis increased significantly (threefold increase, p = 0.002) compared to control rats. On day 4, MTX-treated rats demonstrated a trend toward a decrease in germ cell apoptosis, compared to day 2, and showed histological signs of impaired spermatogenesis (decreased number of germ cell layers and Johnsen’s criteria). A significant increase in cell apoptosis in MTX-treated rats was correlated with higher Bax/Bcl-2 protein levels.

Conclusions

MTX induced germ cell apoptosis and impaired spermatogenesis in rat testes.     

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目的探讨进一步降低大剂量氨甲蝶呤(HDMTX)治疗小儿急性淋巴细胞白血病(ALL)毒副反应的方法。方法对2000年1月至2001年12月在上海儿童医学中心血液肿瘤科住院治疗的47例ALL患儿共进行的134例次HDMTX治疗,按照两种不同的水化、碱化方法,分为全国标准治疗组和观察组。观察患儿血清中的MTX浓度、治疗效果及毒副反应的发生率。结果(1)按WHO相关分度标准,各种毒副反应的发生率、Ⅲ度或Ⅲ度以上的毒副作用者的发生率两组比较差异有显著性意义。(2)观察组有2例次出现尿素氮的升高,标准治疗组未出现。(3)在应用HDMTX开始后44h、68h时血清MTX浓度异常者两组相比较差异有显著性意义。结论观察组HDMTX的毒副反应发生率及严重程度均明显低于全国标准治疗组,且减少治疗费用,而不影响HDMTX治疗效果。     

Pharmacokinetics of HD-MTX in infants,children, and adolescents with non-B acute lymphoblastic leukemia

A retrospective pharmacokinetic analysis was done of methotrexate serum levels after high-dose treatment (HD-MTX, four cycles at two-week intervals of 5 g/sq.¹m. over 24 h i.v.) in children with non-B acute lymphoblastic leukemia (ALL) with the specific aim of seeking differences in patients of different ages, including infants under one year. A total of 122 children (seven infants aged 3 months-1 year, 26 children aged 1–3 years, 68 children aged 3–10 years and 21 adolescents aged 10–15 years) with normal liver and renal function, receiving consolidation therapy at the Pediatric Clinic of Monza between May 1988 and April 1992, were enrolled in this study. MTX was given as an intravenous infusion in 24 h and serum concentrations were measured up to at least 72 h after the start of infusion by an enzyme immunoassay (TDX Abbot, Dallas, TX) in order to modulate folinic acid rescue. Pharmacokinetic analysis of MTX levels according to a two-compartment open model indicated that, compared to all children up to 10 years old, in adolescents older than 10 years the drug reached higher concentrations in serum and was cleared at a lower rate. Steady-state levels and AUC were from 60% higher to more than double and the total clearance of the compound, expressed either per square meter surface area or per kg body weight, in each cycle was significantly lower in adolescents >10 years of age, sometimes being only one-third of the clearance in infants (0.2 vs. 0.6 1/h/kg and 6.6 vs. 10.7 1/h/sq.m). The relationship between each age and systemic clearance was highly significant as measured by regression analysis. Methotrexate systemic clearance progressively decreased as a function of age. Subsequent treatments did not induce changes in MTX pharmacokinetics. These data suggest that the better tolerance of HD-MTX in children may have a pharmacokinetic basis. The faster elimination of MTX in infants, who usually show the worst prognosis, suggests that full doses could be safely used in order to maximize the antileukemic effect without a high risk of toxicity. © 1995 Wiley-Liss, Inc.     

Acral erythema caused by high-dose methotrexate therapy in patients with osteogenic sarcoma

High doses of methotrexate (MTX) have been incorporated in the treatment of osteogenic sarcoma since the 1970s. Unfortunately, high-dose MTX (HD-MTX) can cause untoward side effects that may complicate the proper management of these patients. Two cases of MTX-induced acral erythema are described and the possible implications of this complication are reviewed, with a discussion of the pathophysiology of this adverse effect. Two young female patients suffering from osteogenic sarcoma received HD-MTX (12 g/m²) according to the chemotherapeutic protocol. A 17-year-old patient with osteogenic sarcoma of the head developed painful acral erythema of her palms and soles after the fifth dose of MTX, and a 22-year-old patient with osteosarcoma of the femur developed severe acral erythema after the sixth dose. The severity of the reaction was significant enough to cancel further treatment with HD-MTX in both patients. All symptoms resolved without sequellae several weeks later in both cases. Acral erythema may pose a significant albeit transient phenomenon adversely influencing chemotherapy in patients suffering from osteosarcoma and treated with HD-MTX. The pathophysiology of this side effect is not completely understood and further investigation of the pharmacokinetics of HD-MTX in this patient population is needed.     

High-Dose Methotrexate and Continuous Infusion Ara-C in Children's Non-Hodgkin's Lymphoma: Phase II Studies and Their Use in Further Protocols

Twenty-three children with refractory or relapsed non-Hodgkin's lymphoma (NHL) received high-dose methotrexate (HD-MTX), and 9 received Ara-C by continuous intravenous infusion, as phase II studies. They all had previously received a protocol including vincristine, adriamycin, cyclophosphamide, IV push Ara-C, asparaginase, intrathecal MTX, and cranial irradiation, and had failed to respond or had relapsed. HD-MTX was given at the dose of 6 g/m² or more with leucovorin rescue, Ara-C at the dose of 100 mg/m² /day by continuous infusion over 10 days. Among the 22 evaluable patients receiving HD-MTX, 10 responses (7 CR; 3 PR) were observed. Among the 9 patients receiving Ara-C, 4 responded (1 CR; 3 PR). Toxicity in those previously heavily treated patients was acceptable. These two drugs are now successfully included in childhood NHL treatment protocols.     

High-dose methotrexate therapy of childhood acute lymphoblastic leukemia: lack of relation between serum methotrexate concentration and creatinine clearance.

BACKGROUND: The objectives of this study were: (1) to analyze the relation of serum methotrexate (MTX) concentration with creatinine clearance, (2) to compare the leucovorin rescue dose administered to the patients based on creatinine clearance, with the one calculated according to serum MTX levels, and (3) to determine MTX-related toxicity. PROCEDURE: Thirty children with high-risk non-B acute lymphoblastic leukemia (ALL) treated according to the national protocol (PINDA 92) based on ALL BFM 90, were randomized to receive consolidation with four doses of either 1 or 2 g/m(2) MTX as a 24-hr infusion, at 2-week intervals (group M1 and M2, respectively). Serum MTX concentrations were measured at 24, 42, and 48 hr after beginning the infusion and were analyzed retrospectively. The creatinine clearance was calculated after 12-hr intravenous hydration prior to each MTX dose. Leucovorin dosage was adjusted according to creatinine clearance. RESULTS: Serum MTX concentrations at 24, 42, and 48 hr after starting the infusion were not related to creatinine clearance in both treatment groups. Leucovorin rescue administered according to creatinine clearance was excessive in 43% in group M1 and in 51% in group M2, as compared to the dose calculated according to serum MTX levels. No serious clinical complications were observed. CONCLUSIONS: These results suggest that creatinine clearance is not a good parameter to calculate leucovorin rescue. MTX-related toxicity in this group of patients receiving a dose of 1 or 2 g/m(2) and rescued with leucovorin without monitoring serum MTX levels was acceptable.     

Methotrexate levels and outcome in osteosarcoma

BACKGROUND: Peak serum concentrations of methotrexate (MTX) have been reported to correlate with outcome in osteosarcoma (OS). Modification of the MTX dose to achieve peak levels between 700 and 1,000 micromol/L has been recommended. The goal of the study was to assess whether there is a correlation between histologic necrosis of the tumor and/or prognosis with peak MTX serum concentration. PROCEDURE: Treatment included multi-agent adjuvant chemotherapy, including high-dose MTX (12 g/m2). Peak MTX levels were drawn following a 4-hr infusion. Histologic evaluation for percent necrosis was done at the time of definitive resection. RESULTS: The median peak MTX level (n = 52 patients) was 1,060 micromol/L (range: 410-4,700 micromol/L), with significant intra-patient and inter-patient variability. Fifty-eight percent of the levels were 1,000 micromol/L or higher. Response to pre-operative chemotherapy was: 18% Grade I necrosis, 35% Grade II, 31% Grade III, and 16% Grade IV. No significant association was found between the mean peak MTX levels and necrosis (P = 0.44). Event-free survival (EFS) for the 48 patients with non-metastatic disease at diagnosis was 76% at 4 years of follow-up, with no association between the mean peak MTX level and EFS (P = 0.24). CONCLUSIONS: The absence of a demonstrable correlation between peak MTX levels and histologic necrosis or EFS may suggest that most patients achieve therapeutic levels when MTX is given at a dose of 12 g/m(2). The significant degree of intra-patient variability in peak levels poses a dilemma for pharmacokinetic adjustment. Continued use of HD-MTX in all patients, rather than dose adapted therapy, may be justified.     

Cerebrospinal Fluid and Plasma Methotrexate Levels Following High-Dose Regimen Given as a 3-Hour Intravenous Infusion in Children with Nonhodgkin's Lymphoma

In the French non Hodgkin's lymphoma protocols, central nervous system prophylaxis is provided by high-dose methotrexate (HD-MTX), given as a 3-hour IV infusion of 3 g/m² MTX along with intrathecal MTX injection. The incidence of CNS relapse is less than 3%. We designed a study to evaluate the MTX transfer across the blood brain barrier in terms of cytotoxic concentrations, during these short-term infusions. Cerebrospinal fluid and plasma MTX levels were measured during 61 courses in 29 children with non Hodgkin's lymphoma; none of them had central nervous system disease. Samples were obtained either 4, 12, 18, or 24 hours after the start of HD-MTX IV infusion. A potentially cytotoxic MTX level (10^-6 M) was reached in all courses at 4 hours (median: 2.3 × 10^-6 M) and remained available in 8/16 courses at 12 hours (median: 1.0 × 10^-6 M) and in only 2/17 courses at 18 hours (median: 0.29 × 10^-6 M). Twenty-four hours after the start of HD-MTX IV infusion, CSF MTX level was always less than 10^-6 M. The plasma MTX levels were 260, 1.3, 1.0, and 1.7 × 10^-6 M at 4, 12, 18, and 24 hours, respectively. There was no correlation between plasma and CSF MTX levels. These data show that potentially cytotoxic MTX concentrations can be reached in CSF after a 3-hour IV infusion of 3 g/m² in every patient and remain available for at least 8 hours in half of them.     

Transforming growth factor-alpha stimulates enterocyte proliferation and accelerates intestinal recovery following methotrexate-induced intestinal mucositis in a rat and a cell culture model

Purpose  Recent evidence suggests that transforming growth factor-alpha (TGF-α) enhances enterocyte proliferation and exerts a gut trophic effect. The purpose of the present study was to evaluate the effect of TGF-α on enterocyte proliferation and intestinal recovery following methotrexate (MTX)-induced intestinal mucositis in rats and in Caco-2 cells. Methods  Nonpretreated Caco-2 cells and those pretreated with MTX were incubated with increasing concentrations of TGF-α. Cell proliferation was determined by FACS cytometry. Adult rats were divided into three groups: control rats treated with vehicle, MTX rats treated with one dose (20 μg/kg) of MTX given intraperitoneally, and MTX-TGF-α rats treated with one dose of MTX followed by two doses of TGF-α (75 μg/kg a day). Three days after MTX injection, rats were sacrificed. Intestinal mucosal damage (Park’s score), mucosal structural changes, and enterocyte proliferation were measured at sacrifice. Western blotting was used to determine the level of extracellular signal-related kinase (ERK) protein, a marker of cell proliferation. A nonparametric Kruskal–Wallis ANOVA test was used for statistical analysis with P value less than 0.05 considered statistically significant. Results  The in vitro experiment demonstrated that treatment with TGF-α of Caco-2 cells resulted in a significant stimulation of cell proliferation in a dose-dependent manner. The in vivo experiment showed that treatment with TGF-α resulted in a significant increase in bowel and mucosal weight, DNA and protein content in jejunum and ileum, villus height in jejunum and ileum, crypt depth in ileum, and increased cell proliferation in jejunum and ileum compared to the MTX group. MTX-TGF-α rats also had a significantly lower intestinal injury score in ileum when compared to MTX animals. The increase in levels of cell proliferation in MTX-TGF-α rats corresponded with the increase in ERK protein levels in intestinal mucosa. Conclusion  Treatment with TGF-α prevents mucosal injury, enhances ERK-induced enterocyte proliferation, and improves intestinal recovery following MTX-induced intestinal mucositis in rats. These findings correlated with the observation that TGF-α also caused a significant stimulation of cell proliferation in a Caco-2 cell culture model treated with MTX. These observations may have significant implications for the treatment of patients on chemotherapy who develop severe mucositis.     

Evaluation of overall toxicity of high-dosage methotrexate regimens.

The occurrence of overall toxicity was analyzed for 43 patients with osteosarcoma who received 349 high-dosage courses of methotrexate (HD-MTX) with citrovorum factor (Leukovorin) "rescue" (CF). The dosages of HD-MTX ranged from 50 to 350 mg/kg. Overall toxicity was assessed on the basis of five manifestations of toxicity: stomatitis, dermatitis, myelosuppression, liver dysfunction, and kidney function abnormalities. The great majority (91.4%) of the infusions were well tolerated, but 8.6% were associated with moderate or severe toxicity. Stomatitis and serum glutamic-oxaloacetic transaminase (SGOT) changes were the most frequent postinfusion findings. Three patients died from causes related to MTX toxicity. Dose, age, sex, and number of prior infusions were investigated by logistic regression analysis for prognostic effect on frequency of moderate to severe overall toxicity. Age and number of prior infusions had significant (P less than 0.06) effects on overall toxicity. Patients older than 15 years with greater than 10 prior infusions constituted the "high risk" group with a risk of moderate to severe toxicity 6.3 times that of the younger patients with fewer than 10 infusions.     

胸苷酸合成酶基因多态性对甲氨喋呤治疗急性淋巴细胞白血病患儿不良反应的影响

目的探讨不同胸苷酸合成酶(TS)基因型对急性淋巴细胞白血病(ALL)患儿经大剂量甲氨蝶呤(HD-MTX)治疗后不良反应的影响。方法选取2011年3月至2013年3月确诊的ALL患儿73例,提取其基因组DNA,PCR扩增后测序鉴定TS基因型。观察并记录所有ALL患儿经HD-MTX化疗后的不良反应,并监测化疗后42~48 h MTX血药浓度。结果 73例ALL患儿接受HD-MTX治疗后,其不良反应主要包括中性粒细胞减少、血红蛋白降低、血小板减少、肝脏毒性、黏膜损害和胃肠道反应,不同TS基因型患儿化疗后不良反应发生率比较差异均无统计学意义,各基因型与ALL患儿化疗后42~48 h MTX血药浓度的变化无关联。结论 TS基因多态性对ALL患儿HD-MTX化疗后不良反应的发生无影响。     

Evaluation of overall toxicity of high-dosage methotrexate regimens

The occurrence of overall toxicity was analyzed for 43 patients with osteosarcoma who received 349 high-dosage courses of methotrexate (HD-MTX) with citrovorum factor (Leukovorin) ?rescue”? (CF). The dosages of HD-MTX ranged from 50 to 350 mg/kg. Overall toxicity was assessed on the basis of five manifestations of toxicity: stomatitis, dermatitis, myelosuppression, liver dysfunction, and kidney function abnormalities. The great majority (91.4%) of the infusions were well tolerated, but 8.6% were associated with moderate or severe toxicity. Stomatitis and serum glutamic-oxaloacetic transaminase (SGOT) changes were the most frequent postinfusion findings. Three patients died from causes related to MTX toxicity. Dose, age, sex, and number of prior infusions were investigated by logistic regression analysis for prognostic effect on frequency of moderate to severe overall toxicity. Age and number of prior infusions had significant (P < 0.06) effects on overall toxicity. Patients older than 15 years with greater than 10 prior infusions constituted the ?high risk”? group with a risk of moderate to severe toxicity 6.3 times that of the younger patients with fewer than 10 infusions.     

Risk factors associated with delayed methotrexate clearance and increased toxicity in pediatric patients with osteosarcoma

High‐dose methotrexate (HD‐MTX; 12 g/m²) is part of standard therapy for pediatric osteosarcoma (OS). Risk factors associated with MTX toxicity in children with OS are not well defined. We investigated the association between peak MTX levels (four‐hour) and delayed MTX clearance or treatment toxicity. Information was retrieved from electronic medical records of 33 OS patients treated with HD‐MTX at Texas Children's Hospital from 2008 to 2015. We found that the four‐hour MTX level did not contribute to toxicity or delayed MTX clearance. We demonstrated that certain demographic characteristics are associated with delayed clearance and increased toxicity.     

Modifying Effect of Folinic Acid on Methotrexate-induced Embryotoxicity in Rats

Abstract Embryotoxicity of methotrexate (MTX) and modification of its effect by folinic acid (FA) were evaluated in rats. MTX was administered intraperitoneally to pregnant rats on day 9 of gestation (vaginal plug = day 0), and was followed by an intraperitoneal injection of FA after various time intervals (0-8 hours). Two dose combinations were used; 0.3 mg/kg of MTX and 1.0 mg/kg of FA, and 3.0 mg/kg of MTX and 10.0 mg/kg of FA. The dams were sacrificed on day 20 of gestation, and the fetuses were examined for visceral and skeletal development. The results are as follows: 1) A single dose of 0.3 mg/kg of MTX resulted in high embryolethality and growth retardation in all live fetuses and a single dose of 3.0 mg/kg of MTX showed 100% embryolethality. 2) A single dose of 1.0 or 10.0 mg/kg of FA showed no embryotoxicity. 3) The mitigating effect of FA on MTX-induced embryotoxicity was observed when FA was administered simultaneously with MTX, but was rapidly decreased as the time interval between MTX and FA dosings became longer. 4) Some live fetuses which escaped from MTX embryolethality showed growth retardation and dilation of the cerebral ventricles. The dilation of the cerebral ventricles was found even in the simultaneously treated groups, though the incidences were much lower than the belatedly treated groups.