Toxicological and pharmacologic effects of farnesol (C15H26O): A descriptive systematic review.

The objective of the present study was to perform a systematic review (SR) composed of preclinical and clinical studies which investigated the toxicological and pharmacologic effects of farnesol [Molecular formula: C15H26O; IUPAC: (3,7,11-Trimethyl-2,6,10-dodecatrien-1-ol]. This SR was performed according to PRISMA guidelines. Literature research was performed using PubMed, MEDLINE, Scopus and Web of Science databases using the descriptor combinations: "farnesol and pharmacological effect" and "farnesol and toxicology". The inclusion criteria used were original articles from preclinical and clinical studies investigating the pharmacological and toxicological effects of farnesol, published between January 1960 and December 2017 which were written in English, Portuguese and Spanish. Primary research identified 414 articles, from which 76 articles were selected for final analysis following the inclusion criteria. After grouping, 51.32 and 22.37% of the articles investigated the antimicrobial and antitumor effect, respectively. Methodological biases have been observed both in pre-clinical studies with non-human animals and in clinical trials, mainly in group allocation and blinding. This SR is the first study developed to compile the studies concerning the pharmacological and toxicological effects of farnesol. This study concludes that farnesol possesses different pharmacological and toxicological features, which permit its use as an active or a coadjuvant drug.

Farnesol induces apoptosis-like phenotype in the phytopathogenic fungus Botrytis cinerea.

This study demonstrates that the isoprenoid farnesol produces a toxic effect on the phytopathogenic fungus Botrytis cinerea in solid and liquid media. In solid media farnesol retarded 72 h the beginning of mycelial growth. Also, it was demonstrated that the toxic effect is due to farnesol triggers apoptosis in B. cinerea because ROS accumulation, DNA fragmentation and phosphatidylserine externalization were detected in farnesol-treated mycelium. Therefore, compounds that increase the intracellular farnesol or induce apoptosis could have a potential application as fungicide against B. cinerea.

Farnesol: antinociceptive effect and histopathological analysis of the striatum and hippocampus of mice.

Farnesol, a sesquiterpene alcohol, has been shown to have antioxidant and anti-inflammatory properties. Recent studies have found that antioxidant compounds may exert a certain protective effect against neurotoxicity. The objective of this study was to evaluate the antinociceptive activity of farnesol (FAR) and its neurotoxic effects on the brains of adult mice. In this study, two mouse models of analgesia were used to evaluate FAR at doses of 50, 100, and 200 mg/kg, injected intraperitoneally (i.p.). In the acetic acid-induced writhing test, a significant decrease was found in the number of contortions in the FAR-treated mice at doses of 50, 100, and 200 mg/kg. FAR was also found to inhibit the licking response in the injected paw at doses of 100 and 200 mg/kg (i.p.) in the first (0-5 min) and second phases (15-30 min) of the formalin test. To evaluate neurotoxic effects, Swiss mice were treated with 0.9% saline (i.p., control group), 0.05 Tween 80 dissolved in 0.9% saline (i.p., vehicle group), and FAR 50, 100, or 200 mg/kg, i.p. Following treatment, all groups were observed for 72 h. In the FAR 200-mg group, 16% of the animals suffered brain injury that affected 12% of the area of the hippocampus. No lesions were found in the hippocampal and striatal regions of the brain in any of the animals treated with the 50 and 100 mg/kg doses of FAR. In conclusion, FAR exerts an antinociceptive effect with no significant neurotoxicity in the brains of adult mice.

Evaluation of the mutagenicity of sesquiterpenic compounds and their influence on the susceptibility towards antibiotics of two clinically relevant bacterial strains.

Sesquiterpenic compounds are natural chemicals present in organisms from different Phylae or Divisions, which have proved to be important bioactive products, namely in potentiating the action of antibiotics. In the first step, the mutagenicity of nine sesquiterpenic compounds (hydrocarbons and alcohols) was screened in a Salmonella typhimurium his(-)-reversion test with strains TA98 and TA100, in the presence or absence of in vitro metabolic activation. Under the test conditions, none of the compounds showed mutagenicity up to a concentration of 222µg/plate. trans-Farnesol, nerolidol, and α-bisabolol displayed cytotoxicity when tested at concentrations ranging from 14 to 222µg/plate. Then, the combined effect of antibiotic-sesquiterpenic compounds was evaluated on two clinically relevant pathogens, Escherichia coli and Staphylococcus aureus, with well-defined resistance-sensitive profiles. The agar-disc diffusion assay revealed that all the combinations of antibiotic-sesquiterpenic compounds increased the antibacterial activity of the antibiotics tested against S. aureus. For E. coli, an antagonistic effect was observed for various combinations on the growth of this bacterium.

Microbial quorum-sensing molecules induce acrosome loss and cell death in human spermatozoa.

Infertility in men and women is frequently associated with genital contamination by various commensal or uropathogenic microbes. Since many microorganisms are known to release quorum-sensing signals in substantial amounts, we raised the question whether such molecules can directly affect human spermatozoa. Here we show that farnesol and 3-oxododecanoyl-l-homoserine lactone, employed by the opportunistic pathogenic yeast Candida albicans and the gram-negative bacterium Pseudomonas aeruginosa, respectively, induce multiple damage in spermatozoa. A reduction in the motility of spermatozoa coincided in a dose-dependent manner with apoptosis and necrosis at concentrations which were nondeleterious for dendritic cell-like immune cells. Moreover, sublethal doses of both signaling molecules induced premature loss of the acrosome, a cap-like structure of the sperm head which is essential for fertilization. Addressing their mechanism of action, we found that the bacterial molecule, but not the fungal molecule, actively induced the acrosome reaction via a calcium-dependent mechanism. This work uncovers a new facet in the interaction of microorganisms with human gametes and suggests a putative link between microbial communication systems and host infertility.

Benzo(a)pyrene-induced genotoxicity: attenuation by farnesol in a mouse model.

In the present study we have evaluated the antigenotoxic effects of Farnesol (FL) a 15-carbon isoprenoid alcohol against benzo (a) pyrene [B(a)P] (125 mg kg(- 1).b.wt oral) induced toxicity. B(a)P administration lead to significant induction in Cytochrome P450 (CYP) content and aryl hydrocarbon hydrolase (AHH) activity (p < 0.001), DNA strand breaks and DNA adducts (p < 0.001) formation. FL was shown to suppress the activities of both CYP and AHH (p < 0.005) in modulator groups. FL pretreatment significantly (p < 0.001) restored depleted levels of reduced glutathione (GSH), quinone reductase (QR) and glutathione -S-transferase (GST). A simultaneous significant and at both the doses reduction was seen in DNA strand breaks and in in-vivo DNA adducts formation (p < 0.005), which gives some insight on restoration of DNA integrity. The results support the protective nature of FL. Hence present data supports FL as a future drug to preclude B (a) P induced toxicity.

Fragrance material review on farnesol.

A toxicologic and dermatologic review of farnesol when used as a fragrance ingredient is presented.

A chemogenomic screen in Saccharomyces cerevisiae uncovers a primary role for the mitochondria in farnesol toxicity and its regulation by the Pkc1 pathway.

The isoprenoid farnesol has been shown to preferentially induce apoptosis in cancerous cells; however, the mode of action of farnesol-induced death is not established. We used chemogenomic profiling using Saccharomyces cerevisiae to probe the core cellular processes targeted by farnesol. This screen revealed 48 genes whose inactivation increased sensitivity to farnesol. The gene set indicated a role for the generation of oxygen radicals by the Rieske iron-sulfur component of complex III of the electron transport chain as a major mediator of farnesol-induced cell death. Consistent with this, loss of mitochondrial DNA, which abolishes electron transport, resulted in robust resistance to farnesol. A genomic interaction map predicted interconnectedness between the Pkc1 signaling pathway and farnesol sensitivity via regulation of the generation of reactive oxygen species. Consistent with this prediction (i) Pkc1, Bck1, and Mkk1 relocalized to the mitochondria upon farnesol addition, (ii) inactivation of the only non-essential and non-redundant member of the Pkc1 signaling pathway, BCK1, resulted in farnesol sensitivity, and (iii) expression of activated alleles of PKC1, BCK1, and MKK1 increased resistance to farnesol and hydrogen peroxide. Sensitivity to farnesol was not affected by the presence of the osmostabilizer sorbitol nor did farnesol affect phosphorylation of the ultimate Pkc1-responsive kinase responsible for controlling the cell wall integrity pathway, Slt2. The data indicate that the generation of reactive oxygen species by the electron transport chain is a primary mechanism by which farnesol kills cells. The Pkc1 signaling pathway regulates farnesol-mediated cell death through management of the generation of reactive oxygen species.

Modulation of hepatic and renal drug metabolizing enzyme activities in rats by subchronic administration of farnesol.

Farnesol demonstrates antitumor activity in several animal models for human cancer and was being considered for development as a cancer chemopreventive agent. This study was performed to characterize the effects of minimally toxic doses of farnesol on the activity of phase I and II drug metabolizing enzymes. CD((R)) rats (20/sex/group) received daily gavage exposure to farnesol doses of 0, 500, or 1000 mg/kg/day for 28 days; 10 rats/sex/group were necropsied at the termination of farnesol exposure; remaining animals were necropsied after a 28-day recovery period. No deaths occurred during the study, and farnesol had no significant effects on body weight, food consumption, clinical signs, or hematology/coagulation parameters. Modest but statistically significant alterations in several clinical chemistry parameters were observed at the termination of farnesol exposure; all clinical pathology effects were reversed during the recovery period. At the termination of dosing, the activities of CYP1A, CYP2A1-3, CYP2B1/2, CYP2C11/12, CYP2E1, CYP3A1/2, CYP4A1-3, CYP19, glutathione reductase, NADPH/quinone oxidoreductase and UDP-glucuronosyltransferase were significantly increased in the livers of farnesol-treated rats; farnesol also increased the activity of glutathione S-transferase in the kidney. The effects of farnesol on hepatic and renal enzymes were reversed during the recovery period. At the end of the dosing period, increases in absolute and relative liver and kidney weights were seen in farnesol-treated rats. These increases may be secondary to induction of drug metabolizing enzymes, since organ weight increases were not associated with histopathologic alterations and were reversed upon discontinuation of farnesol exposure. Administration of farnesol at doses of up to 1000 mg/kg/day induced reversible increases in the activities of several hepatic and renal drug metabolizing enzymes in rats, while inducing only minimal toxicity. It is concluded that non-toxic or minimally toxic doses of farnesol could alter the metabolism, efficacy, and/or toxicity of drugs with which it is co-administered.

The antibacterial effects of terpene alcohols on Staphylococcus aureus and their mode of action.

The study was made of the antibacterial effects of three terpene alcohols on Staphylococcus aureus, focusing on the leakage of K+ ions and toxicity over time. The leakage of K+ ions was monitored continuously with a K+-electrode. Our results suggested that the terpene alcohols, namely, farnesol, nerolidol and plaunotol might act on cell membranes. The rank order of effectiveness, farnesol>nerolidol>plaunotol, was the same in the toxicity assay and in the examination of the leakage of K+ ions, when we considered the initial rate and the amount of leaked K+ ions. The rank order agreed with the results of a growth-inhibition assay reported previously. The antibacterial activity reflected the initial rate of leakage of K+ ions, suggesting that damage to cell membranes might be one of the major modes of action of these terpene alcohols. The results also demonstrated that the initial rate of leakage and the amount of leaked K+ ions are useful as indices of the antibacterial activities of hydrophobic compounds.

The novel Ras antagonist, farnesylthiosalicylate, suppresses growth of prostate cancer in vitro.

BACKGROUND: The majority of men with advanced prostate cancer (PCa) respond to androgen deprivation therapy (ADT) with objective evidence of tumor regression. However, these tumors will regrow in the presence of low-androgen levels after 12-18 months. Regrowth after ADT is associated with upregulation of growth factor (GF) mediated pathways. The compound farnesylthiosalicylate (FTS), a specific antagonist of the 21 kDa Ras protein, suppresses GF signaling and it might be a useful therapy against advanced PCa. METHODS: We measured androgen and GF dependent growth of androgen dependent LNCaP and androgen hypersensitive CWR-R1 PCa cells in response to specific inhibitors of GF pathways, including FTS. Inhibition of GF mediated signaling and cell-cycle pathways was confirmed by Western blotting of extracts from treated cells. RESULTS: Both LNCaP and CWR-R1 cells were dependent on GF signaling pathways for cell growth. FTS, as well as suppressing cell growth, inhibited GF signaling pathway activity and reduced the levels of E2F1, p-Rb, and p-cdc2, all GF dependent mediators of cell-cycle progression. CONCLUSIONS: These data suggest that FTS might be a useful agent against PCa that has relapsed after ADT.

Farnesyl thiosalicylic acid chemosensitizes human melanoma in vivo.

Malignant melanoma is well known for its poor response to a variety of chemotherapeutic agents. Testing of numerous treatment strategies has identified dacarbazine as the most active single drug; however, its response rates in various clinical settings are quite limited. Defective apoptosis in combination with oncogenic proteins (such as activated Ras) in cell proliferation pathways plays a key part in both the development and disease progression of human melanoma. Farnesyl thiosalicylic acid, a novel Ras inhibitor, dislodges Ras proteins from the cell membrane, leading to inhibition of cell transformation and tumor growth. In this study we evaluated the effect of farnesyl thiosalicylic acid treatment on established human melanoma xenografts grown in mice with severe combined immunodeficiency as well as the chemosensitizing effect of farnesyl thiosalicylic acid in combination with dacarbazine. Daily administration of 10, 20, or 40 mg per kg of farnesyl thiosalicylic acid resulted in a concentration-dependent reduction in tumor growth, with growth inhibition reaching a mean value of 45+/-7%, at the highest concentration. The combination of farnesyl thiosalicylic acid (10 mg per kg per day) and dacarbazine (80 mg per kg per day) resulted in a significant reduction of 56%+/-9%, in mean tumor growth. Analysis of toxicologic parameters (mouse weight, blood cell counts, and blood chemistry) showed an acceptable and similar toxicity profile for both the single-agent farnesyl thiosalicylic acid treatment and the combination of farnesyl thiosalicylic acid plus dacarbazine treatment. Given the observed preclinical treatment responses and the low toxicity, our results support the notion that farnesyl thiosalicylic acid in combination with dacarbazine may qualify as a rational treatment approach for human melanoma.

A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice.

Constitutively active Ras proteins, their regulatory components, and overexpressed tyrosine kinase receptors that activate Ras, are frequently associated with cell transformation in human tumors. This suggests that functional Ras antagonists may have anti-tumor activity. Studies in rodent fibroblasts have shown that S-trans, transfarnesylthiosalicylic acid (FTS) acts as a rather specific nontoxic Ras antagonist, dislodging Ras from its membrane anchorage domains and accelerating its degradation. FTS is not a farnesyltransferase inhibitor, and does not affect Ras maturation. Here we demonstrate that FTS also acts as a functional Ras antagonist in human pancreatic cell lines that express activated K-Ras (Panc-1 and MiaPaCa-2). In Panc-1 cells, FTS at a concentration of 25-100 microM reduced the amount of Ras in a dose-dependent manner and interfered with serum-dependent and epidermal growth factor-stimulated ERK activation, thus inhibiting both anchorage-dependent and anchorage-independent growth of Panc-1 cells in vitro. FTS also inhibited tumor growth in Panc-1 xenografted nude mice, apparently without systemic toxicity. Daily FTS treatment (5 mg/kg intraperitoneally) in mice with tumors (mean volume 0.07 cm3) markedly decreased tumor growth (after treatment for 18 days, tumor volume had increased by only 23+/-30-fold in the FTS-treated group and by 127+/-66-fold in controls). These findings suggest that FTS represents a new class of functional Ras antagonists with potential therapeutic value.

Selective cytotoxicity of farnesylamine to pancreatic carcinoma cells and Ki-ras-transformed fibroblasts.

Farnesyl protein transferase (FPTase) catalyses the post-translational modification of proteins by a farnesyl pyrophosphate. One of the substrates of this enzyme is p21ras, the product of the ras oncogene. We examined whether farnesylamine, one of the FPTase inhibitors (FTI), is selectively cytotoxic in pancreatic carcinoma cells and Ki-ras-transformed fibroblasts. Furthermore, we investigated whether the cytotoxicity of farnesylamine is caused by the induction of apoptosis in these cells. Using the FPTase assay, we found that farnesylamine inhibited FPTase in vitro. Immunoprecipitation showed that farnesylamine inhibited farnesylation of p21ras in vivo. In addition, 24 and 5 microM farnesylamine were required to achieve 50% cytotoxicity in pancreatic carcinoma cells containing activated Ki-ras and Ki-ras-transformed NIH/3T3 cells, respectively. The parental NIH/3T3 cells were resistant to the cytotoxic effect of farnesylamine at concentrations less than 100 microM. After incubation with farnesylamine, DNA fragmentation was observed in both pancreatic carcinoma cells and Ki-ras-transformed fibroblasts at cytotoxic doses of this compound but not in NIH/3T3 cells. These results indicate that the mechanism of cell death induced by farnesylamine is apoptosis, and this apoptosis occurred specifically in pancreatic carcinoma cells containing mutated Ki-ras and the Ki-ras-transformed cells. Because raf is downstream of ras (p21ras) in the ras-raf-mitogen-activated protein kinase signaling pathway, we used c-raf-1-transformed fibroblasts, which proved to be resistant to apoptosis induced by farnesylamine. This supports the theory that inhibition of ras signaling may be related to the induction of apoptosis. These data further suggest that farnesylamine could be useful as a chemotherapeutic agent in cancers that very frequently contain a Ki-ras oncogene mutation, e.g., pancreatic cancer.

Selective farnesol toxicity and translocation of protein kinase C in neoplastic HeLa-S3K and non-neoplastic CF-3 cells.

We have reported earlier that farnesol, a 15 carbon isoprenoid, has inhibitory effects on the growth and viability of a variety of cultured cells of neoplastic derivation but is considerably less cytotoxic to cells derived from normal tissue (Cancer Lett., 79, 175-179). As part of our search for the mechanism of this observation, we have studied the effect of 20 microM farnesol on the distribution of protein kinase C (PKC) between cytosolic and membrane fractions of HeLa S3K cells and fibroblasts line CF-3. In HeLa cells farnesol caused translocation of PKC from membrane fraction to cytosol after 1h of incubation and also prevented PMA-stimulated induction of PKC translocation from cytosol to membranes. Up to 6 h of incubation, there was no effect of farnesol on PKC localization in CF-3 fibroblasts. The results point to possible involvement of PKC in the toxic effect of farnesol which occurs with some degree of selectivity depending on cell origin.

[A 13-week subcutaneous toxicity study of prednisolone farnesylate (PNF) in rats].

The toxicity of Prednisolone farnesylate (PNF), a synthetic glucocorticoid, was investigated in the Sprague-Dawley rat. PNF was injected subcutaneously at doses of 0.03, 0.3, 3 and 30 mg/kg/day for 13 weeks. In addition, 18.7 mg/kg/day prednisolone (PN), which is approximate to 30 mg/kg/day PNF in prednisolone molarity, was also administered to the rat for comparison. The results are summarized as follows: 1. All animals from the PN 18.7 mg/kg/day group, and four(4) out of ten(10) males and three(3) out of ten(10) females from the PNF 30 mg/kg/day group died having shown weakened condition such as unkempt fur and emaciation. Histopathologically, systemic suppurative inflammation, as shown by pyeronephritis and abscess formation in many organs and tissues, was observed and it was considered that the administration of steroid induced weakened condition and systemic suppuration which resulted in death. In addition, atrophy was noted in the adrenal glands, lymphatic organs and skin, and histopathological lesions were also observed in the lungs, liver, pancreatic islets, bone, bone marrow and mammary glands. 2. Surviving animals in the PNF 30 mg/kg/day group showed almost the same changes as those observed in the dead animals that died. Hematological examination revealed an anemic change and a decrease in lymphocytes with an increase in segmented neutrophils and eosinophils. In the urinalysis and blood chemistry, the changes suggesting damages to the liver and kidneys were mainly observed. 3. In the PNF 3 and 0.3 mg/kg/day groups, several changes such as atrophy of the adrenal glands, lymphatic organs and skin were noted in a dose dependent manner. 4. In the PNF 0.03 mg/kg/day group, ther were no toxic signs. 5. Based on these results, it was concluded that the overt toxic dose of PNF was 0.3 mg/kg/day and the non-toxic dose was 0.03 mg/kg/day in the present study.

[Reproductive and developmental toxicity study of prednisolone farnesylate (PNF)--study by subcutaneous administration of PNF prior to and in the early stages of pregnancy in rats].

A fertility study of Prednisolone farnesylate (PNF), a newly synthesized corticosteroid, was conducted in Sprague-Dawley rats. This compound was administered subcutaneously at dose levels of 0(control), 0.04, 0.2 and 1 mg/kg/day to males for 63 days before mating and during the mating period, and to females for 14 days before mating, through the mating period and until day 7 of pregnancy. Each 24 male and female rats were mated, and females were killed on day 20 of pregnancy to examine their fetuses. 1. In the parental animals, loss of fur or thin fur and incrustation of treated site occurred in male rats treated at doses of 0.2 mg/kg or more and female rats treated at dose of 1 mg/kg, and at the same dose groups, the thinning of skin, atrophy of the thymus and intention of the substance at the injected site were noted. Moreover, body weight gains and food consumption were suppressed in both sexes treated at the dose of 1 mg/kg. 2. Fertility and reproductive ability in both sexes, and estrus cycles in female rats were not affected by administration of PNF. 3. In the fetuses, no embryonic or fetal lethal effect and teratogenic effect were noted. From these results, the no-effect dose levels of PNF on the parental general states, the parental reproductive ability and those of the fetuses are thought to be 0.04 mg/kg/day, 1 mg/kg/day or more and 1 mg/kg/day or more, respectively, under the experimental conditions of this study.

[A 13-week percutaneous toxicity study of prednisolone farnesylate (PNF) gel in beagle dogs with a recovery period of 5 weeks].

The toxicity of Prednisolone farnesylate (PNF) gel, a synthetic glucocorticoid, was investigated in the Beagle dog. PNF gel was administered percutaneously at doses of 0.2, 0.8 and 3.2 mg/kg/day for 13 weeks, then the drug was withdrawn for 5 weeks to evaluate the reversibility. In addition, 2 mg/kg/day prednisolone gel (PN gel), which is approximate to 3.2 mg/kg/day PNF gel in prednisolone molarity, was also administered for comparison. The results are summarized as follows: 1. No deaths were observed in any of the PNF gel test groups or the PN gel group, nor were there any abnormal findings in the clinical signs of the animals. 2. In the hematology, a tendency toward a decrease in the lymphocyte ratio was observed in males from the PNF gel 0.8 mg/kg/day and above groups. In the PN gel group, a significant decrease or a tendency toward a decrease in the lymphocyte ratio was observed, as well as an increase in the white blood cell count in some animals. 3. In the blood biochemistry, a significant decrease or a tendency toward a decrease in total cholesterol and phospholipid was observed in males from the PNF gel 3.2 mg/kg/day group and a tendency toward an increase in triglyceride in females from the PNF gel 3.2 mg/kg/day group was observed. In the PN gel group, a tendency toward an increase in AIP activity, a tendency toward an increase in triglyceride were observed. 4. In the histological examinations, a decrease in the weight of the thymus and adrenal glands, vacuolation of hepatocytes in the middle zone of the liver, atrophy of zona fasciculata of the adrenal glands, hypertrophy of zona glomeruli, swelling of cortical cells of zona faciculate and atrophy of the thymus were observed in the PNF gel 0.8 mg/kg/day and above groups. In the PN gel group, atrophy of submandibular lymph nodes and mesenteric lymph nodes was observed in addition to the same changes as observed in the PNF gel groups. Furthermore, thinning, atrophy or a decrease in the weight of the adrenal glands was also observed both in the PNF gel 3.2 mg/kg/day group and the PN gel group at the end of the 5-week recovery period. 5. As described above, a decrease in the lymphocyte ratio, in the weight of the thymus and adrenal glands and vacuolation of hepatocytes were observed in the PNF gel 0.8 mg/kg/day and above groups.(ABSTRACT TRUNCATED AT 400 WORDS)

[A 52-week percutaneous toxicity study of prednisolone farnesylate (PNF) gel in beagle dogs with a recovery period of 8 weeks].

The toxicity of Prednisolone farnesylate (PNF) gel, a synthetic glucocorticoid, was investigated in the Beagle dog. PNF gel was administered percutaneously at doses of 0.05, 0.2 and 0.8 mg/kg/day for 52 weeks, then the drug was with held for 8 weeks to evaluate reversibility. The results are summarized in the following. 1. In the 0.05 mg/kg/day and above groups, hypotrichosis in the application site of the skin, thinning of the skin and atrophy of the appendages, and in the 0.2 mg/kg/day and above groups a tendency toward retarded body weight gain, were observed. 2. In the 0.2 mg/kg/day and above groups, a drop in the lymphocyte ratio, a rise in GOT activity and A1P level, and in the 0.8 mg/kg/day group a rise in free fatty acid were observed. 3. In the 0.2 mg/kg/day group and above groups, atrophy of the zona fasciculata and zona reticularis were observed. In the 0.8 mg/kg/day group, a decrease in the weight of the thymus and adrenal glands, and a increase in the weight of the liver, were observed. 4. At the end of the recovery period, most of the changes disappeared, except for those in the adrenals and treated area. From the above results, under the conditions of this study, it was concluded that when the changes observed in the application site of the skin in each group were not taken into consideration, the toxicological no effect level was 0.05 mg/kg/day for both males and females and the overt toxic dose level was 0.8 mg/kg/day.