Procurement and Characterization

Preparation and Analysis of Dose Formulations

The dose formulations were prepared by mixing diisopropylcarbodiimide and anhydrous ethanol to give the required concentrations; formulations were prepared once not more than 7 days prior to the study start and every 3 weeks thereafter for the 20-week study or monthly for the 27-week study. The dose formulations were stored at room temperature for the 20-week study and the 27-week study until April 6, 1999, or later, when formulations were stored at −20° C for up to 35 days.

Because the dose formulations were true solutions of the test article in ethanol, homogeneity studies were not performed. Prior to the 20-week study, a stability study of 2.19 mg/mL dose formulations of lot 01207BG was conducted by the study laboratory using GC; stability was confirmed for up to 35 days for the dose formulation stored at ambient temperature in sealed containers under a nitrogen headspace and for up to 3 hours when exposed to light and air at ambient temperature.

Periodic analyses of the dose formulations of diisopropylcarbodiimide were conducted by the study laboratories using GC. During the 20-week study, the dose formulations were analyzed four times; animal room samples of these dose formulations were also analyzed. All 20 dose formulations analyzed were within 10% of the target concentrations; all 20 of the animal room samples analyzed were within 10% of the target concentrations. Dose formulations were analyzed four times during the 27-week study; animal room samples of these dose formulations were also analyzed. Of the 20 dose formulations analyzed, all were within 10% of the target concentrations; 9 of 20 animal room samples were within 10% of the target concentrations. Unusual degradation was observed in the animal room samples from the formulations prepared on February 17, 1999, and June 7, 1999. Attempts to discover the cause of the degradation in the formulations were not conclusive, but the most likely cause, water in the anhydrous ethanol, was eliminated. To prevent degradation of the test chemical, formulations prepared on or after April 6, 1999, were stored at −20° C.

20-Week Study

Female FVB/N-TgN(v-Ha-ras)Led (Tg.AC) hemizygous mice were obtained from Taconic Farms, Inc. (Germantown, NY). On receipt, the mice were 4 weeks old. Animals were quarantined for 13 days and were 6 weeks old on the first day of the study. Before the study began, five mice were randomly selected for parasite evaluation and gross observation for evidence of disease. Blood was collected from five randomly selected vehicle control mice at study termination. The sera were analyzed for antibody titers to rodent viruses (Boorman et al., 1986; Rao et al., 1989a,b); all results were negative.

The dose levels selected for this study were based on results from a 3-month toxicity study in B6C3F₁ mice (NTP, 2006). Groups of 10 male and 10 female mice were dermally administered 0, 17.5, 35, 70, 140, or 280 mg/kg diisopropylcarbodiimide 5 days per week for 3 months. All 280 mg/kg males and females and nine 140 mg/kg males and females died before the end of the study. The final body weight gain of 70 mg/kg males was significantly less than that of vehicle controls. At the site of application, there were significant increases in the incidences of epidermal hyperplasia in males and females administered 70 mg/kg or greater, chronic inflammation in 140 and 280 mg/kg males and 70 mg/kg or greater females, and sebaceous gland hyperplasia in 140 mg/kg males. Based on these results, 70 mg/kg was selected as the high dose for the current studies with four lower doses of 35, 17.5, 8.75, and 4.38 mg/kg.

Groups of 10 female mice received dermal applications of 0, 4.38, 8.75, 17.5, 35, or 70 mg/kg in ethanol, 5 days per week for 20 weeks. A dosing volume of 2 mL ethanol per kg body weight was applied to the center of a shaved dorsal area extending from the posterior of the scapulae to the base of the tail, with the application site no greater than 10% of the animal’s body surface. A minimum of two consecutive days of dosing were performed prior to terminal sacrifice, with the last dose within 24 hours of sacrifice. Animals were housed individually, with feed and water available ad libitum. Body weights were recorded initially, weekly, and at the end of the study, and clinical findings were recorded weekly. Details of the study design and animal maintenance are summarized in Table 1.

Table 1

Experimental Design and Materials and Methods in the Dermal Studies of Diisopropylcarbodiimide.

Necropsies were performed on all animals. The heart, right kidney, liver, lung, and thymus were weighed. Microscopic examinations were performed on all vehicle control and 70 mg/kg animals and all animals that died early. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 µm, and stained with hematoxylin and eosin. Table 1 lists the tissues and organs routinely examined.

27-Week Study

Female B6.129-Trp53^tm1Brd (N5) haploinsufficient mice were obtained from Taconic Farms, Inc. (Germantown, NY). On receipt, the animals were approximately 5 weeks old. Animals were quarantined for 13 days and were 7 weeks old on the first day of the study. Before initiation of the study, five mice were randomly selected for parasite evaluation and gross observation for evidence of disease. Blood was collected at study termination from five randomly selected vehicle control mice. The sera were analyzed for antibody titers to rodent viruses; all results were negative.

The dose levels selected for this study were based on results from a 3-month toxicity study in B6C3F₁ mice (NTP, 2006) as described for the 20-week study. Groups of 15 female mice received dermal applications of 0, 4.38, 8.75, 17.5, 35, or 70 mg/kg in ethanol 5 days per week for 27 weeks. The dosing volume of 2 mL/kg was applied to the center of a shaved dorsal area posterior of the scapulae to the base of the tail, with the application site no greater than 10% of the animal’s body surface. A minimum of two consecutive days of dosing were performed prior to terminal sacrifice, with the last dose given within 24 hours of sacrifice. Mice were housed individually, with feed and water available ad libitum. Body weights were recorded initially, weekly, and at the end of the study, and clinical findings were recorded weekly. Details of the study design and animal maintenance are summarized in Table 1.

Necropsies were performed on all animals. Histopathologic examinations were performed on all mice. Skin samples from the site of application were cut in an anterior-posterior direction, trimmed or marked with indelible ink to indicate the anterior end, and placed on an index card to keep them flattened during fixation. Control skin samples from an untreated area were also taken. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 µm, and stained with hematoxylin and eosin. Table 1 lists the tissues and organs routinely examined.

Upon completion of the laboratory pathologist’s histopathologic evaluation, the slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit. The slides, individual animal data records, and pathology tables were sent to an independent pathology laboratory where quality assessment was performed. Results were reviewed and evaluated by the NTP Pathology Working Group (PWG); the final diagnoses represent a consensus of contractor pathologists and the PWG. Details of these review procedures have been described by Maronpot and Boorman (1982) and Boorman et al. (1985).

Statistical Methods

Quality Assurance Methods

The 20- and 27-week studies were conducted in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58). In addition, as records from these studies were submitted to the NTP Archives, these studies were audited retrospectively by an independent quality assurance contractor. Separate audits covered completeness and accuracy of the pathology data, pathology specimens, final pathology tables, and a draft of this NTP Report. Audit procedures and findings are presented in the reports and are on file at NIEHS. The audit findings were reviewed and assessed by NTP staff, and all comments were resolved or otherwise addressed during the preparation of this Report.