Sensitization potential of gold sodium thiosulfate in mice and guinea pigs.

Since gold sodium thiosulfate (GST) has been included in a standard patch test series for diagnosis of allergic contact dermatitis from gold, the incidence of patients showing positive reactions to gold is increasing. However, there were little reports on induction of gold sensitization in animals. In this study, we have examined the sensitization potential of GST using mice and guinea pigs. In the guinea pig maximization test, 2 or 6 out of 10 animals showed positive skin responses, mainly edema, by challenge with 2% or 5% GST in 50% ethanol solution, respectively. In the mouse ear swelling test, positive ear swelling (20% greater increase in ear thickness) after challenge with GST was shown in 2 out of 6 mice those previously treated with GST. Topical exposure of mice to GST in 70% dimethylsulfoxide solution induced small increases in the lymph node weight and the lymph node cell (LNC) number in the murine local lymph node assay (LLNA). A greater degree of LNC responses were observed in the sensitive mouse lymph node assay (SLNA) compared with the LLNA, but the stimulation index of total lymph node response by GST was not so high. From these results, GST was identified as a contact allergen, but the sensitization potential was not so strong. In the mouse IgE test, treatment of mice with GST resulted in a statistically significant increase in the serum IgE antibody concentration that associated with immediate-type hypersensitivity reaction. It may suggest that the sensitization responses from gold would appear not only at the contact site but also systematically.

Allergic contact dermatitis from a pyridine derivative in polyvinyl chloride leather.

Antimicrobial coating of household products has gained wide acceptance in Japan in the past several years. Pyridine derivatives, used as antifungal or antibacterial agents in many common products, are known to cause contact dermatitis. We present a case of severe contact dermatitis caused by a pyridine derivative used as an antifungal agent in the polyvinyl chloride (PVC) leather of a chair. An open patch test was performed with each ingredient of the PVC leather. Other products were previously eliminated from consideration based on a series of negative patch tests. The PVC leather obtained from the patient's chair gave a ++ reaction with evident blistering, according to the International Contact Dermatitis Research Group standard. Fifteen ingredients of the PVC leather were open patch tested; a positive reaction was found with 2,3,5,6-tetrachloro-4 (methylsulphonyl) pyridine (1% in petrolatum). Clinicians should be aware that antifungal or antibacterial agents may be increasingly incorporated into common household products and should be suspected in cases of contact dermatitis.

[Determination of antimicrobial, 10,10'-oxybis-10H-phenoxarsine, in artificial leather used for the seat of chairs].

Recently, a case of patients with allergic contact dermatitis caused by the poly (vinyl chloride) (PVC) seat, containing 10,10'-oxybis-10H-phenoxarsine (OBPA), of a chair was reported. OBPA was developed as an antimicrobial for plastics such as PVC and polyurethane, and it has been widely used in artificial leather for the seats of chairs and sofas. To identify causative chemicals for allergic contact dermatitis, a combination of patch testing in the patients and chemical analysis of causative products is valuable. However, no analytical method and data of OBPA in commercial products was reported. In this study, a method for the determination of OBPA in the artificial leather (PVV base) was developed. OBPA was extracted from PVC samples with methanol. The extract was loaded on an aluminum oxide column, and washed with diethyl ether:hexane, and eluted with ethanol:hexane. The eluate was evaporated, dissolved in methanol and injected to a HPLC equipped an ODS column and an UV detector (detection wavelength 300 nm). OBPA standard crystal was isolated from commercial agents containing OBPA. The calibration curve for OBPA was linear in the range of 0.1-100 micrograms/ml. The minimum detection and determination concentrations of OBPA in samples were 0.07 and 0.25 microgram/g. By this method, eight PVC sheets for the seat of a chair were analyzed. In two PVC sheets, 52.7 and 84.9 micrograms/g of OBPA were detected. In the PVC product that caused contact dermatitis, OBPA was not found.

Activation of osteoblast-like MC3T3-E1 cell responses by poly(lactide).

This study examined the osteoblast-like MC3T3-E1 cell responses to poly(DL-lactide) (PDLLA) and poly(L-lactide) (PLLA) with different weight average molecular weight (M.W.). Colony formation of MC3T3-E1 cells on the PLLA with M.W. 270000 or 1370000 was slightly lower than that on glass. The protein, DNA and hydroxyproline (HYP) content and alkaline phosphatase (ALP) activity for cells cultured on the PLLA (M.W. 270000 or 1370000) for 14 d were almost similar to those on glass. In contrast, the ALP activity of the cells cultured on low M.W. PLLA (M.W. 20000) increased. Osteoblast differentiation was stimulated by low M.W. PLLA but not by high M.W. PLLA. The addition of low M.W. PDLLA (M.W. 5000 or 10000), L-lactide or L-lactic acid into culture increased the protein, DNA and HYP content and ALP activity for cells at 100 microg/ml. Compared with four chemicals, PDLLA (M.W. 10000) had the strongest simulation effect on the cell. The release of L-lactic acid from PLLA and PDLLA into aqueous solution during incubation only slightly affected cell activity. In a cell-free condition, in the presence of PDLLA, the ALP activity was maintained without inactivation, even after 24 h incubation. Such a phenomenon was not seen with L-lactide and L-lactic acid. This may be a reason why PDLLA has a stronger effect on osteoblast differentiation relative to L-lactic acid. These results suggested that increased osteoblast differentiation was induced by low M.W. PDLLA and PLLA, and these may be used as a effective material in the field of orthopedic and drug delivery systems for the treatment of bone diseases.

Allergic contact dermatitis from a perinone-type dye C.I. Solvent Orange 60 in spectacle frames.

This is the 1st case report of allergic contact dermatitis from a perinone-type plastic dye, C.I. Solvent Orange 60, used in the earpieces of spectacle frames. Sensitization of this dye was confirmed by patch tests and chemical analysis of the causative earpieces and coloring agents. Solvent Orange 60 is suspected of being the contact allergen in at least 2 other Japanese cases of spectacle earpiece dermatitis, and provoked strong reactions on sensitized individuals. Its use in products that are applied on human skin for a prolonged period of time, such as spectacle frames or hearing aids, would best be avoided.

Contact dermatitis due to rubber boots worn by Japanese farmers, with special attention to 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (ETMDQ) sensitivity.

An investigation was conducted as follows in 9 farmers with contact allergy due to rubber boots: (i) patch tests with 19 rubber additives; (ii) chemical analysis of additives in 6 pairs of rubber boots; (iii) use tests on a hypoallergenic trial product in 5 patients. The following results were obtained: (i) in the patch tests, all 9 patients showed positive reactions to 1 or more of the nitrogen(N)-containing antioxidants (IPPD, DMBPPD, ETMDQ); (ii) ETMDQ was detected in 1 pair of rubber boots, and IPPD and DMBPPD in another pair; (iii) no patient using hypoallergenic boots during rice-planting had recurrent dermatitis. N-containing antioxidants, such as IPPD, DMBPPD and ETMDQ, were thus considered as the main causative agents and the trial product was found useful for managing contact dermatitis. Contact allergy due to ETMDQ in rubber is reported here for the 1st time.

[Twenty-eight days repeated dose toxicity test of N-(fluorodichloromethylthio)phthalimide in rats].

N-(Fluorodichloromethylthio)phthalimide (Fluor-folpet) has been widely used as an anti-mold and anti-bacterial agent. In this study, 28 days repeated-dose oral toxicity study of fluor-folpet was carried out in Slc:Wistar rats. An oral toxicity study for fluor-folpet, the twenty-eight days test, repeated-dose, oral administration, was performed as follows: Five week-old rats, male and female, 10 rats, each/group, were treated with intragastric administration of fluor-folpet with a dose of 0 (1% Sodium CMC, control), 20, 80 and 320 mg/kg, body weight. Recovery test, for 14 days after the last treatment, was examined for the control and the 320 mg/kg groups. The 320 mg/kg groups, both males and females, showed significantly reduced their body-weight gain compared with the control group. In the 320 mg/kg group, five out of 20 male rats and four out of 20 female rats died from dyspnea during the treatment period. In the female rats in the 320 mg/kg group, serum ChE level was decreased to 50% of control level and gamma-GT was increased in a dose-dependent manner, but these serum levels recovered after 14 days non-treatment period. No histopathological change, relating to the treatment, in liver was observed. Increased weight of the kidney and vacuolation in renal tubules were found in both sexes of 320 mg/kg group. Hyperkeratosis and hyperplasia of the stomach epithelium were observed at the dose more than 80 mg/kg in male, and more than 20 mg/kg in female. A supplemental study, repeated-dose, oral administration in rats carried out to examine the dyspnea revealed that severe acute toxic damages in epithelium of nasal cavity and meatus nasopharyngeus were induced by intragastric administration of fluor-folpet. Fluor-folpet is shown to be cytotoxic. In conclusion, the no-observed-effect level (NOEL) for fluor-folpet was not found under the experimental conditions employed in this repeated-dose toxicity study.

[Comparative studies on a single dose toxicity of microsomal Ca(2+)ATPase inhibitor, 2,5-di(tert-butyl)-1,4-hydroquinone and its related analog, mono(tert-butyl)-1,4-hydroquinone, in rats].

We performed comparative studies to determine an acute toxicity of microsomal Ca(2+)ATPase inhibitor, 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) and its related analog, mono(tert-butyl)-1,4-hydroquinone (MTBHQ), which are both used as antioxodants. Wistar rats, 5 weeks old, male and female, were used. By a single dose of oral administration, DTBHQ-induced LD50 values (obtained by Lorke method) in male and female rats were estimated 295.1 and 234.4 mg/kg BW, respectively, whereas each LD50 value for MTBHQ was 711.6 and 400.0 mg/kg BW, respectively. MTBHQ-induced deaths occurred from 8 to 20 minutes after administration, however, DTBHQ-induced deaths occurred more delayed from 1 to 5 days after administration. The observed toxic signs of DTBHQ included diarrhea (jelly like), prone position, lacrimation, salivation and abnormal gait (such as reluctance to walk, limping). Localized purpura and loss of the tail (perhaps as a result of necrosis) were also observed. In comparison, MTBHQ elicited prone position, panting, staggering gait and spastic gait. Without loss of the tail montioned above, dead and sacrified rats showed no remarkable changes in macroscopic examination due to exposure to both compounds.

Identification of causative chemicals of allergic contact dermatitis using a combination of patch testing in patients and chemical analysis. Application to cases from rubber gloves.

5 cases of allergic contact dermatitis from rubber gloves were investigated by our recommended procedures using a combination of patch testing in patients and chemical analysis of causative rubber products by gas chromatography (GC) and high-performance liquid chromatography (HPLC). We previously confirmed that zinc ethylphenyldithiocarbamate (ZEPC), a dithiocarbamae-type accelerator (DTC), was causative in a case of allergic contact dermatitis from rubber work gloves. Subsequently, we have clarified that DTCs such as zinc dimethyldithiocarbamate (ZDMC), zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) and amines such as dimethylamine (DMA), diethylamine (DEA) and piperidine (PIP) were also causative in cases from surgical rubber gloves. Thus, our investigative studies revealed that, although thiurams have been taken much more notice of as allergenic compounds than their corresponding DTCs and amines, not only DTCs such as ZDMC, ZDEC, ZDBC and ZEPC, but also amines such as DMA, DEA and PIP were noteworthy causative candidates of allergic contact dermatitis from rubber gloves.

A new protocol and criteria for quantitative determination of sensitization potencies of chemicals by guinea pig maximization test.

This paper presents precise sensitization test data of 15 chemicals with a wide spectrum of sensitization potencies, and proposes a new protocol and criteria for quantitative evaluation of sensitization potencies of chemicals. The tests were performed according to the design of Magnusson and Kligman, changing the application concentrations for induction as well as for challenge phases. 3-dimensional relationships between mean response (or sensitization rate), induction and challenge concentrations were found in all chemicals tested. The following 2 values are proposed as a quantitative measure of sensitization potency: (a) the minimum induction concentration that induces a positive response; (b) the challenge concentration that induces a mean response approximately equal to 1.0 among the animals applied with the highest concentration for induction. Both values coincided with each other within the range of 1 order of magnitude in every compound except 2. The values varied by 5 orders or more of magnitude among the compounds, showing a wide variation of sensitization potencies among chemicals. A good correlation was found for every chemical between the value of sensitization potency thus obtained and the residual levels in causative products in human cases of allergic contact dermatitis. A new experimental protocol for obtaining values (a) and (b) is proposed.

[Subchronic toxicity study of tributoxyethyl phosphate in Wistar rats].

Tributoxyethyl phosphate (TBEP) is widely used in household materials such as plasticizer, floor polish and flame retardant in plastic resins and synthetic rubbers. This compound has been detected at ppb level in underground water. In order to elucidate the toxicity of TBEP, a 14-week oral toxicity study was conducted. Wistar rats (5-weeks old, male & female, 15 rats/group) were given diet containing 0, 0.03, 0.3 or 3.0% TBEP. Suppression of body weight gain was observed in both sexes of the 3.0% group. Serum cholinesterase activity was significantly decreased in both sexes of the 0.3 and 3.0% groups and serum gamma-glutamyl transferase activity was significantly increased in both sexes of the 3.0% group after 5 and 14 weeks exposure. Amylase in serum was also increased in 0.3 and 3.0% group males and 3.0% group females. Absolute and relative liver weights in both sexes were significantly increased in the 3.0% group after 5 and 14 weeks of exposure. Histopathological examination revealed moderate periportal hepatocyte swelling in male rats of the 3.0% group after 14 weeks exposure but this change was not found in male rats given 0.3% or less of TBEP in the diet or in any of the females. These findings indicated that the liver is a target organ for TBEP toxicity. We concluded a no-observed effect level (NOEL) of TBEP in the diet of 0.03% (male: 20 mg/kg/day, female: 22 mg/kg/day) under the conditions of this toxicity study.

Identification of causative chemicals of allergic contact dermatitis using a combination of patch testing in patients and chemical analysis. Application to cases from industrial rubber products.

3 cases of allergic contact dermatitis from industrial rubber products were investigated using a combination of patch testing in patients and chemical analysis of causative rubber products by gas chromatography (GC), GC-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Our studies revealed N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), a typical rubber allergen, as a causative chemical in a case from a brand of heavy-duty rubber gloves and a case from a black rubber ring for car manufacture, and zinc ethylphenyldithiocarbamate (ZEPC), a dithiocarbamate (DTC)-type accelerator, as a causative chemical in a case from a brand of rubber work gloves: both IPPD and ZEPC, which showed positive patch test reactions, were confirmed in the extracts of the causative rubber products by GC, GC-MS and/or HPLC.

Identification of causative chemicals of allergic contact dermatitis using a combination of patch testing in patients and chemical analysis. Application to cases from rubber footwear.

5 cases of allergic contact dermatitis from rubber footwear were investigated by a combination of patch testing in patients and chemical analysis of causative rubber products. Our studies revealed 2-mercaptobenzothiazole (MBT) and benzothiazyl disulfide (MBTS) (typical allergenic accelerators) as causative chemicals in 3 cases from children's rubber shoes, ladies' rubber boots and ladies' canvas shoes. These 3 patients reacted to mercaptobenzothiazole-type accelerators including MBT and MBTS. MBT and MBTS were determined in each item of causative footwear by chemical analysis, including extraction by shaking with acetone-chloroform (1:1) mixture at room temperature and determination using reversed-phase high-performance liquid chromatography (RP-HPLC). Subsequently, we identified styrenated phenol (SP), a newly found allergenic antioxidant, as a causative chemical in a case from ladies' canvas shoes. The patient reacted to SP but not to MBT and MBTS, though SP, MBT and MBTS were determined in the causative shoes by gas chromatography (GC), GC-mass spectrometry (GC-MS) and HPLC. We also identified p-tert-butylphenol-formaldehyde resin (PTBP-F-R), (a known allergenic adhesive ingredient) as a causative chemical in a case from ladies' sneakers. The patient reacted to PTBP-F-R but not to p-tert-butylphenol (PTBP), MBT and MBTS. These 4 compounds were determined in the causative sneakers by GC, GC-MS and HPLC. Thus, our studies revealed that not only known allergens, such as MBT, MBTS and PTBP-F-R, but also a newly found one, such as SP, were important causes of allergic contact dermatitis from rubber footwear.

[Dermatological evaluation of a flame retardant, hexabromocyclododecane (HBCD) on guinea pig by using the primary irritation, sensitization, phototoxicity and photosensitization of skin].

As one of the projects in the safety evaluation of chemical constituents in common house-hold products, effects of hexabromocyclododecane (HBCD) were evaluated by primary skin irritation, skin sensitization, phototoxicity and photosensitization using guinea pigs. Primary skin irritation was not observed in HBCD emulsified in distilled water by the Draize test method. Skin sensitization test was carried out according to the maximization test method of Magnusson and Kligman. For this test, HBCD was dissolved in olive oil to give 5, 0.5 and 0.05%. When induction of sensitization occurred, challenged doses of 0.005, 0.05, 0.5 and 5% of HBCD (dissolved in acetone) were applied to its respective sensitized groups. The results showed that the induction dose of greater than 0.5% and the challenge dose of greater than 0.05% elicited a positive response. The increase in the concentration of induction and challenge doses did not further increase the percentage of positive response or the intensity of skin response. Phototoxicity test was carried out with 0, 0.5 and 5% of HBCD dissolved in acetone. Phototoxicity was not observed at all HBCD concentration tested. Photosensitization test was performed according to the Sato's adjuvant-strip method. The skin sensitization and challenge reaction doses were 5 and 0.5% and 0 and 0.5% HBCD (dissolved in acetone), respectively, and no positive reaction was observed. It is clear from the foregoing results that HBCD is a mild skin allergen.

A method for identifying causative chemicals of allergic contact dermatitis using a combination of chemical analysis and patch testing in patients and animal groups: application to a case of rubber boot dermatitis.

A 63-year-old woman developed allergic contact dermatitis from rubber boots. Initial investigation, by patch testing in the patient and chemical analysis of the causative rubber boots, revealed that mercaptobenzothiazole (MBT) and dibenzothiazyl disulfide (MBTS) were the causative chemicals. Subsequent investigations were performed by patch testing in animal groups. An extract of the causative rubber boots, MBT and MBTS were used for sensitization of guinea pigs by the guinea pig maximization test (GPMT). 3 animal groups, A (with the boot extract), B (with MBT) and C (with MBTS) were successfully prepared. The boot extract was fractionated by column chromatography and thin-layer chromatography (TLC). Each fraction was subjected to patch testing in the animal groups. Positive reactions in all groups would show that the active fractions contained MBT-type compounds, whereas a positive reaction in group A but negative ones in group B and C would show that the active fractions did not contain any MBT-type compounds. Each fraction was then analyzed by gas chromatography (GC), GC-mass spectrometry (GC-MS), direct inlet-MS (DI-MS) and high-performance liquid chromatography (HPLC). By this investigation, we found not only known allergens (MBT, MBTS), but also unknown allergens: S-substituted MBT-type compounds and styrenated phenol (SP). Thus, SP was shown to be a candidate as a human sensitizer even though the patient did not react to it.

Tinuvin P in a spandex tape as a cause of clothing dermatitis.

We report a 54-year-old female with allergic contact dermatitis from 2-(2-hydroxy-5-methylphenyl)benzotriazole (Tinuvin P) in a spandex tape sewn into underwear. Tinuvin P is used as an ultraviolet light absorber for stabilizing plastics and the other organic materials against discoloration and deterioration. It is effective in protecting synthetic and natural fibers, polyesters, chlorinated polyesters, polystyrene, polyvinyls, cellulose acetate, ethyl cellulose, acrylates, dyes, waxes, detergents, cosmetic formulations, etc. Our patient developed itchy erythema on her shoulders, chest and upper back after wearing underwear for 1 night. She showed positive reactions to spandex tape sewn into the underwear. She also reacted to 2-(2-hydroxy-5-methylphenyl)benzotriazole (Tinuvin P), which was contained in the spandex. No cross-reaction to other benzotriazoles was seen.

Comparative studies by cell culture and in vivo implantation test on the toxicity of natural rubber latex materials.

Colony assay using V79 cells, the agar diffusion assay with L929 cells, and the 7-day rabbit muscle implantation test were employed to evaluate the cytotoxicity and tissue toxicity of natural rubber latex (NRL) materials. The in vivo implantation test showed that, among 13 histological parameters, thickness of inflammatory layer was the most useful index to evaluate tissue responses quantitatively. A comparison of the in vivo and in vitro parameters revealed the following correlations between the thickness of the inflammatory layer and cytotoxicity indices: Colony assay of the extracts, IC50: r = 0.80; Agar diffusion assay, Zone index: r = 0.73; Lysis index: r = 0.61. From these results, it appears that the colony assay provides a more reliable prediction of the tissue response than the agar diffusion assay.