Administration Of Test Materials Research Articles

Implications for room lighting and the duration of acclimation protocols on the dosimetry of inhaled drugs in rats

Inhaled administration of test materials to animals has technical challenges for quantitative dosimetry. Inhaled “doses” reported in non-clinical studies are often calculated using an equation such as that of Alexander et al. [1] to estimate the respired minute volume (eRMV) from body weight data. This approach may overlook physiological effects on lung function associated with a formulation’s properties or habituation of animals to the “dosing” technique. Rats are insensitive to red light [2] and anecdotal data (unpublished) suggested red lighting may induce a calmer state in restrained rats. We hypothesized that the lighting color (red or white) and duration of restraint tube acclimation protocols may influence the breath frequency and minute volume of rats during inhalation exposure, representing a potential source of variability in achieved doses. Head-out plethysmography [3] was used to investigate this hypothesis concurrent with inhaled administration of an anti-inflammatory drug.

Further studies on membrane stabilizing, anti-inflammatory and FCA induced arthritic activity of various fractions of bark of Machilus macrantha in rats

Machilus macrantha Nees, Lauraceae, bark is traditionally used in the treatment of asthma, tuberculosis and rheumatoid arthritis. In order to validate, mechanism based anti-inflammatory activity of fractions M. macrantha bark are investigated for first time. Test materials viz. petroleum ether (PE), alkaloidal fraction (CH), acetone extracts (TAN) and mucilage (MM) (250 and 500 mg/kg, p.o.) obtained from M. macrantha bark were tested for membrane stabilizing, anti-nociceptive; anti inflammatory and Freund's complete adjuvant (FCA) induced arthritis activity. Diclofenac sodium and morphine were used as the reference standards in pharmacological assay. Test materials have significantly (p<0.01) inhibited paw edema after Carrageenan and histamine induction at higher doses. Administration of test materials of M. macrantha (250 and 500 mg/kg b.w.) significantly reduced abdominal writhing, formalin nociception, cotton pellet granuloma and vascular permeability in experimental animal. In addition to this, bark of M. macrantha showed chronic anti-rheumatic effect by suppressing the swelling volume, arthritis index, hematological and biochemical parameters (ESR, RA factor, CRP, liver transferase enzyme) in FCA-induced arthritis. It also significantly inhibited protein denaturation, heat-induced haemolysis of RBC and reduction in total leukocyte migration. Bioassay guided fractionation of the pet. ether extract of bark of M. macrantha led to isolation and characterization of β-sitosterol and stigma sterol confirmed by its HPLC, NMR and GC-MS study. In conclusion, extracts of M. macrantha bark can be explored as a therapeutic agent for the treatment of acute and chronic arthritis.

The effect of tannic acid and polyethylene glycol on the absorption capacity of chicken intestine for d‐xylose and β‐carotene

In an experiment, the possible influence of tannic acid (TA) and polyethylene glycol (PEG) on the absorption capacity of intestine for d-xylose and β-carotene in broiler chicken was investigated. Four groups of nine 28-day-old broiler cockerels received d-xylose (500 mg) and β-carotene (52 μg) solutions (Group 1 to 4) with TA (1 g, Group 2 to 4) and PEG (500 mg Group 3 and 1 g Group 4), orally. One blood sample prior to, and four others after the administration of test materials, were collected from wing vein on 40 min basis, for 160 min and the concentration of plasma d-xylose was determined. The concentration of β-carotene was also measured in plasma of blood samples taken prior to and 160 min post-administration of the test materials. Plasma d-xylose concentration of all groups showed quadratic correlations with time (p < 0.001, r(2) = 0.84, 0.60, 0.70 and 0.74 for Group 1 to 4, respectively). Administration of TA reduced the plasma concentration of d-xylose in Group 2. However, feeding PEG after TA raised the concentration of d-xylose in Group 4 to the level that there was no difference in that variable between this group and Group 1. Although the plasma concentration of β-carotene was increased in 160 min post-ingestion of the test material, no difference was found in that variable among the experimental groups. In conclusion, TA and its interaction with PEG have impacts on the absorption capacity of intestine for d-xylose and highly likely other simple sugars, but TA or PEG have no influence on the absorption of β-carotene and most probably other fat soluble vitamins.

Absorption capacity of chicken intestine for d-xylose in response to graded concentrations of tannic acid

Dietary tannins alter the mechanisms involved in cellular uptake and transport of simple sugars. An experiment was conducted on broiler chickens to evaluate the absorptive response of intestine to the graded concentrations of tannic acid (TA), using d-xylose absorption test. Four groups of ten 28-d-old broiler cockerels, were given d-xylose solution alone (500 mg kg −1 BW, group 1) and with TA solution at a dose of 0.5, 1.0 and 1.5 g/bird (groups 2–4, respectively), orally. One blood samples prior-to, and four others after administration of test material, were collected by ulnar vein puncture on 40 min basis for 160 min, and the concentration of d-xylose in plasma was measured. The plasma d-xylose concentration in each group had a quadratic correlation with time ( P<0.001, R 2 = 0.75, 0.84, 0.73 and 0.84 for groups 1–4, respectively). Tannic acid delayed and severely suppressed the intestinal absorption of d-xylose in groups 2–4 ( P<0.001). The plasma d-xylose concentration of group was negatively related to the level of dosed TA with an exponential trend at each time of blood sampling ( R 2 = 0.88, 0.87, 0.86 and 0.83 for 40, 80, 120 and 160 min post-ingestion of TA solution, respectively). Tannic acid showed to be a strong luminal inhibitor of sugar absorption. However, the ingestion of higher concentrations of TA (1.5 g/bird) did not further reduce the concentration of plasma d-xylose suggesting the influence of TA on the intestinal absorption capacity was dose-dependent in an exponential manner.

Immunoadjuvant potential of Asparagus racemosus aqueous extract in experimental system

The immunoadjuvant potential of Asparagus racemosus (Willd.) Family (Liliaceae) aqueous root extract was evaluated in experimental animals immunized with diphtheria, tetanus, pertussis (DTP) vaccine. Immunostimulation was evaluated using serological and hematological parameters. Oral administration of test material at 100 mg/kg per day dose for 15 days resulted significant increase (P=0.0052) in antibody titers to Bordtella pertussis as compared to untreated (control) animals. Immunized animals (treated and untreated) were challenged with B. pertussis 18323 strain and the animals were observed for 14 days. Results indicate that the treated animals did show significant increase in antibody titers as compared to untreated animals after challenge (P=0.002). Immunoprotection against intra-cerebral challenge of live B. pertussis cells was evaluated based on degree of sickness, paralysis and subsequent death. Reduced mortality accompanied with overall improved health status was observed in treated animals after intra-cerebral challenge of B. pertussis indicating development of protective immune response. Present study indicates applications of test material as potential immunoadjuvant that also offers direct therapeutic benefits resulting in less morbidity and mortality.

Immunoadjuvant potential of Asparagus racemosus aqueous extract in experimental system

The immunoadjuvant potential of Asparagus racemosus (Willd.) Family (Liliaceae) aqueous root extract was evaluated in experimental animals immunized with diphtheria, tetanus, pertussis (DTP) vaccine. Immunostimulation was evaluated using serological and hematological parameters. Oral administration of test material at 100 mg/kg per day dose for 15 days resulted significant increase ( P=0.0052) in antibody titers to Bordtella pertussis as compared to untreated (control) animals. Immunized animals (treated and untreated) were challenged with B. pertussis 18323 strain and the animals were observed for 14 days. Results indicate that the treated animals did show significant increase in antibody titers as compared to untreated animals after challenge ( P=0.002). Immunoprotection against intra-cerebral challenge of live B. pertussis cells was evaluated based on degree of sickness, paralysis and subsequent death. Reduced mortality accompanied with overall improved health status was observed in treated animals after intra-cerebral challenge of B. pertussis indicating development of protective immune response. Present study indicates applications of test material as potential immunoadjuvant that also offers direct therapeutic benefits resulting in less morbidity and mortality.

IgG and IgE antibody responses following exposure of Brown Norway rats to trimellitic anhydride: comparison of inhalation and topical exposure

A variety of chemicals can cause sensitisation of the respiratory tract and occupational asthma, including certain acid anhydrides, diisocyanates and reactive dyes. As yet, no well-validated methods are available for the toxicological evaluation of the respiratory sensitising potential of chemicals. One approach which has been explored recently is the evaluation of induced IgE responses or cytokine expression patterns in rats or mice following topical exposure to chemical. Thus, it has been demonstrated that topical exposure of rodents to respiratory sensitising chemicals, but not to contact allergens, causes a dose-dependent and time-related increase in the concentration of total IgE. Using the reference respiratory allergen trimellitic anhydride (TMA), we have considered here the influence of route of exposure on the nature of induced immune responses. Specific IgG and IgE antibody responses and changes in total serum concentration of IgE have been measured following exposure of Brown Norway (BN) starin rats to TMA by topical administration or by inhalation. Exposure to TMA by both routes resulted in the stimulation of specific IgG and IgE antibody, although responses were considerably more vigorous after dermal exposure. Topical treatment also provoked marked and sustained increases in total serum IgE levels, whereas exposure via the respiratory tract stimulated a more transient elevation of this immunoglobulin in a minority of animals which reached statistical significance only at the highest dose group. The lesser vigour of the immune response following inhalation exposure is likely to be related to the considerably lower total antigenic dose which is delivered by this route. Nevertheless, these results show that the nature of immune response with respect to antibody isotype profile provoked by topical administration of TMA is qualitatively comparable with that stimulated by inhalation exposure to the same chemical. For the purposes of hazard assessment and identification of potential chemical respiratory allergens as a function of induced changes in serum IgE concentration, however, the evidence is that topical administration of test material is the preferred route of exposure.

Improved chromium-cobalt-molybdenum alloy for orthopaedic applications : F.Duflos, M.Pierronnet. (Tecphy, Imphy, France.)

Wear-debris powders of cobalt-chromium-molybdenum (CoCrMo) and titanium-aluminum-vanadium (TiAlV) alloys, which are widely used for orthopedic implants (eg, hip and knee prostheses), were tested for carcinogenic activity following intraarticular administration (20 mg/rat) to groups of 44 male Fischer-344 rats (Charles River Breeding Laboratories, North Wilmington, MA). Control groups received similar intraarticular injections of either a noncarcinogen (manganese powder, negative control rats) or a potent carcinogen (nickel subsulfide powder, positive control rats). The experimental groups of 8–12 rats were observed for 24 months after injection. No local tumors developed at the injection site in the negative control rats or in rats that received the CoCrMo or TiAlV powders; poorly differentiated or pleomorphic sarcomas developed at the injection site in 10 of the 12 positive control rats that were treated with nickel subsulfide. Incidences of primary tumors distant from the injection site did not differ significantly among the experimental groups. This study shows that, under experimental conditions, any carcinogenic activity of CoCrMo or TiAlV wear-debris powders is weak in comparison to nickel subsulfide. Based on this study and observations in other laboratories, intraarticular administration of test materials to rats provides a practical, reliable, and biologically relevant method for carcinogenesis testing of biomaterials used for orthopedic implants.

Assessment of the chorioallantoic membrane vascular assay (CAMVA) in the COLIPA in vitro eye irritation validation study

The chorioallantoic membrane vascular assay (CAMVA) is an alternative to the Draize rabbit eye irritation method. The CAMVA employs the vascularized membrane of a fertile hen's egg to assess eye irritation potential. This irritation potential is a function of alterations in the vasculature following the administration of test material. Because of the history of use of the CAMVA it was selected as one of the methods for a validation study organized and sponsored by COLIPA. For this validation study mathematical prediction models (PMs) were developed to convert the CAMVA results into predicted Draize eye irritation scores known as a modified maximum average Draize score (MMAS). These predicted scores were statistically compared with the observed scores to assess the relevance of the CAMVA. The assay was conducted on the same set of test materials by two independent laboratories. These two sets of data were compared to assess the interlaboratory reproducibility of the assay. The results of this validation study of the CAMVA show that for test materials with MMASs in the 0 to 5 range or the 55 to 110 range, the CAMVA did not give a good prediction. The predictions were better for samples of mild to moderate irritation (MMAS 5–55). The difficulty in predicting at the low end of the irritation scale appears to be due to the biological variability of the test system and the subjective nature of the CAMVA evaluation. For those samples with an MMAS above 55, the CAMVA appeared to be limited in demonstrating the more severe response. This may be due to the fact that the PMs were developed using historical data sets of test materials with MMASs below this range. Two approaches for improving the CAMVA for eye irritation prediction are (1) to decrease the variability at the low end by reducing the subjectivity in the scoring and (2) to develop better prediction models using more data in the range of severe irritants.

Intraarticular carcinogenesis bioassays of CoCrMo and TiAlV alloys in rats

Wear-debris powders of cobalt-chromium-molybdenum (CoCrMo) and titanium-aluminum-vanadium (TiAlV) alloys, which are widely used for orthopedic implants (eg, hip and knee prostheses), were tested for carcinogenic activity following intraarticular administration (20 mg/rat) to groups of 44 male Fischer-344 rats (Charles River Breeding Laboratories, North Wilmington, MA). Control groups received similar intraarticular injections of either a noncarcinogen (manganese powder, negative control rats) or a potent carcinogen (nickel subsulfide powder, positive control rats). The experimental groups of 8–12 rats were observed for 24 months after injection. No local tumors developed at the injection site in the negative control rats or in rats that received the CoCrMo or TiAlV powders; poorly differentiated or pleomorphic sarcomas developed at the injection site in 10 of the 12 positive control rats that were treated with nickel subsulfide. Incidences of primary tumors distant from the injection site did not differ significantly among the experimental groups. This study shows that, under experimental conditions, any carcinogenic activity of CoCrMo or TiAlV wear-debris powders is weak in comparison to nickel subsulfide. Based on this study and observations in other laboratories, intraarticular administration of test materials to rats provides a practical, reliable, and biologically relevant method for carcinogenesis testing of biomaterials used for orthopedic implants.

Effects of Bran, Lignin and Deoxycholic Acid on the Permeability of the Rat Cecum and Colon

The ability of dietary fiber to modify the effects of a bile acid on permeability of the cecum and colon was studied. A cecal catheter, which permits administration of test materials to conscious, unrestrained rats over a period of several weeks, was designed. Rats were fed fiber-free diet or diets containing 20% bran or lignin. Permeability of the lower intestine was assessed indirectly by infusing polyethylene glycol (PEG) and measuring excretion of PEG in urine. Under these conditions there was little effect of diet on permeability of PEG. However, when sodium deoxycholate was infused with the PEG, permeability was increased in rats fed fiber-free and diets. In contrast, rats fed the bran diet showed no such response to the bile acid. The interaction among type of dietary fiber, presence of bile acid and intestinal permeability may have important implications for the etiology of intestinal disease.

Betel quid chewing and oral cancer: Experimental studies on hamsters

Betel quid ingredients--betel nut, betel leaf, lime, catechu and tobacco--were tested separately and in various combinations for carcinogenicity, using hamster cheek pouch as the experimental site. The four modes of administration used were (1) tri-weekly painting of the cheek pouch with aqueous extracts of test materials, (2) deposition of replaceable wax pellets containing the test material, (3) gelatin capsules containing the powdered material and (4) insertion of natural material into the pouch for trauma and direct exposure. Untreated controls and standard carcinogen DMBA-treated controls were also maintained. A total of 317 young adult golden Syrian hamsters (Mesocricetus auratus) used for the experiments were killed in two age groups: 6-12 months and 13-24 months, only when signs of general debility were observed. In the untreated controls, animals were free of any malignancy. In the experimental series, various betel quid ingredient combinations under test induced both oral and gastric lesions ranging from massive atypia and precancerous lesions to frank carcinomas. Maximum lesions were observed in the groups receiving betel nut, lime and tobacco combinations and in the polyphenol fraction of betel nut containing major tannins. The mode of administration of test material resulted in distinct differences; tri-weekly paintings giving oral lesions in the range of 22-23% and gastric lesions 39-48%; the same material given either through the replaceable gelatin capsule or in natural form induced 69% oral lesions and 63 to 82% gastric lesions. Overall evaluation of the data of all the four series confirms the potent carcinogenicity of betel nut, particularly its tannin-containing polyphenolic fraction and its combination with lime and tobacco. Maximum oral lesions induced in the hamsters by continuous exposure to capsules and natural material, highlight the direct relationship of frequency of chewing in habitual chewers with oral carcinogenesis. The high incidence of gastric (forestomach) lesions invites special attention.

Clinical studies on ACTH in the blood. I. Blood ACTH levels in normal subjects and in various endocrine diseases; effect of various steroids

ACTH was assayed by the measurement of corticosterone in the adrenal venous plasma after the intravenous administration of test materials into the hypophysectomized rats whose right adrenal glands had been extirpated one week before. This method gave linearity of response to log doses of ACTH ranging from 0.03 to 1.0 milliunits (mU), with precision index (λ) of 0.32, enabling to detect 0.6 mU per 100 ml of plasma ACTH when a dose of 5 ml of plasma was injected.1) Plasma from 5 normal subjects was found to contain ACTH in levels less than 0.6 mU per 100 ml. The oxycellulose technic was applied to extract ACTH from about 100 ml blood from 5 normal subjects in preparation for a biological assay, which revealed a mean ACTH level of 0.04 mU per 100 ml of blood. (96% fiducial range, 0.023-0.063 mU/100 ml of blood.) Four patients with Cushing's syndrome due to adrenal cortical hyperplasia and 2 patients with the same syndrome due to adrenal cortical adenoma failed to show elevated plasma ACTH levels. Similarly, no ACTH was detected in 7 patients with abnormal pigmentation and 9 of 10 patients with hyperthyroidism. Seven patients with adrenogenital syndrome had elevated ACTH levels ranging from 0.6 to 5.7 mU per 100 ml of plasma (mean±standard error (S.E.), 1.6±0.8 mU/100 ml of plasma.) In 10 patients with Addison's disease, plasma ACTH ranged from 0.9 to 93.8 mU per 100 ml (mean±S.E., 13.8±9.5 mU/100 ml of plasma.) 2) The elevated level of ACTH found in patients with adrenogenital syndrome and Addison's disease was significantly reduced by the oral administration of glucocorticoids, whereas aldosterone and desoxycorticosterone had no significant suppresive effect. One of the anabolic steroids, i.e. 2-hydroxymethylene-17α-methyl dihydrotestosterone (HMD), and testosterone had no effect on plasma ACTH levels, either.