Irritancy and allergic responses induced by topical application of ortho-phthalaldehyde

Abstract

Although ortho-phthalaldehyde (OPA) has been suggested as an alternative to glutaraldehyde for the sterilization and disinfection of hospital equipment, the toxicity has not been thoroughly investigated. The purpose of these studies was to evaluate the irritancy and sensitization potential of OPA. The EpiDerm Skin Irritation Test was used to evaluate in vitro irritancy potential of OPA and glutaraldehyde. Treatment with 0.4125 and 0.55% OPA induced irritation, while glutaraldehyde exposure at these concentrations did not. Consistent with the in vitro results, OPA induced irritancy, evaluated by ear swelling, when mice were treated with 0.75%. Initial evaluation of the sensitization potential was conducted using the local lymph node assay at concentrations ranging from 0.005 to 0.75%. A concentration-dependent increase in lymphocyte proliferation was observed with a calculated EC3 value of 0.051% compared to that of 0.089%, previously determined for glutaraldehyde. Immunoglobulin (Ig) E-inducing potential was evaluated by phenotypic analysis of draining lymph node (DLN) cells and measurement of total and specific serum IgE levels. The 0.1 and 0.75% exposed groups yielded significant increases in the IgE+B220+ cell population in the lymph nodes while the 0.75% treated group demonstrated significant increases in total IgE, OPA-specific IgE, and OPA-specific IgG(1). In addition, significant increases in interleukin-4 messenger RNA and protein expression in the DLNs were observed in OPA-treated groups. The results demonstrate the dermal irritancy and allergic potential of OPA and raise concern about the proposed/intended use of OPA as a safe alternative to glutaraldehyde.

FIG. 1.

Irritancy potential following treatment with OPA or glutaraldehyde. Bars represent the mean percent viability ± SE of six EpiDerm tissues determined by MTT assay. “*” Indicates significant difference (p < 0.01) between treatment group and untreated control. “#” Indicates significant difference (p < 0.01) between OPA and glutaraldehyde at indicated concentration.

FIG. 2.

Ear swelling as a result of topical application of OPA. Analysis of irritation after topical application of OPA. Bars represent mean ± SE of five mice (10 ears) per group. Levels of statistical significance are denoted as “*” (p < 0.01) as compared to DMF vehicle.

FIG. 3.

Allergic sensitization potential after dermal exposure to OPA. Analysis of the allergic sensitization potential of OPA using the LLNA. ³H-thymidine incorporation into DLN cells of BALB/c mice following exposure to vehicle or concentration of OPA. Numbers in boxes appearing above the bars represent the SI for each concentration tested. Bars represent mean ± SE of five mice per group. Levels of statistical significance are denoted as “*” (p < 0.05) and “**” (p < 0.01) as compared to DMF vehicle.

FIG. 4.

Serum total IgE levels after dermal exposure to OPA. Analysis of total IgE levels in mouse sera after dermal exposure to OPA. Bars represent mean fold change ± SE of five mice per group. Levels of statistical significance are denoted as ** (p < 0.01) as compared to DMF vehicle. Dermal treatment with 0.75% glutaraldehyde produced 206 ± 26 ng/ml of IgE (p < 0.05).

FIG. 5.

Serum levels of antibodies specific for OPA protein adducts. Analysis of serum antibodies (A, IgE; B, IgG₁; and C, IgG_2a) specific for OPA-MSA conjugates determined using indirect ELISA. Samples were considered to be positive if the OD was 10 times that of the DMF control. For mice treated with 0.75% OPA, five out of five, three out of five, and three out of five mice were positive for OPA-MSA IgG₁, IgG_2a, and IgE, respectively. Bars represent the mean fold change ± SE of five mice per group. The heat treated-0.75 group represents serum from 0.75% OPA group that was heat treated to destroy IgE while leaving IgG intact. Significant difference at p < 0.05 relative to *DMF control group and “#” respective MSA antibody titer.

FIG. 6.

DLN gene expression measured by quantitative real-time PCR. Quantitative real-time PCR analysis of IL-4 (A) or IFN-γ expression (B). Bars represent mean fold change ± SE of five mice per group. Levels of statistical significance are denoted as “*” (p < 0.01) as compared to DMF vehicle.

FIG. 7.

DLN IL-4 protein expression. Analysis of IL-4 protein expression generated by stimulated DLN after dermal exposure to OPA. Bars represent mean fold change ± SE of five mice per group. Levels of statistical significance are denoted as “*” (p < 0.05) and “**” (p < 0.01) as compared to DMF vehicle. Dermal treatment with 0.75% glutaraldehyde generated 217 ± 46 pg/ml of IL-4 in the DLN (p < 0.01).